Lapachone delivery systems, compositions and uses related thereto

ABSTRACT

In part, the present invention is directed to a system comprising a lapachone or a prodrug thereof and a polymer, such as a biocompatible and optionally biodegradable polymer, methods for treatment using the subject polymer compositions, and methods of making and using the same. In another part, the present invention includes inclusion complexes of a lapachone or a prodrug thereof and a cyclodextrin, preferably a β-cyclodextrin, such as hydroxypropyl β-cyclodextrin, e.g., to improve the solubility of the lapachone or prodrug thereof.

RELATED APPLICATION

This application claims the benefit of, and incorporates by reference,the entire disclsoure of U.S. Provisional Patent Application No.60/374693, filed Apr. 23, 2002.

BACKGROUND OF THE INVENTION

β-Lapachone (β-lap) is a potent cytotoxic agent that demonstratesantitumor activity against a variety of human cancer cells atconcentrations typically in the range of 1-10 μM (IC₅₀). The drug wasfirst isolated from the bark of the Lapacho tree (genus Tabebuia) in therainforests of South America, which has a long history as an herbalmedicine. Cytotoxicity has been demonstrated in transformed cell linesderived from patients with promyelocytic leukemia, prostate, malignantglioma, hepatoma, colon, breast, ovarian, pancreatic, and multiplemyeloma cell lines including drug-resistant lines. (Planchon et al.,Cancer Res., 55 (1996) 3706; Li, C. J., et al., Cancer Res., 55 (1995)3712; Weller, M. et al., Int. J Cancer, 73 (1997) 707; Lai, C. C., etal. Histol Histopathol, 13 (I 998) 8; Huang, L., et al., Mol Med, 5,(1999) 711; Wuertzberger, S. M., et al., Cancer Res., 58 (1998) 1876;Li, C. J. et al., Proc. Natl. Acad Sci. USA, 96(23) (1999) 13369-74; Li,Y., et al., Mol Med, 6 (2000) 1008; and Li, Y. Z., Mol Med, 5 (1999)232). No cytotoxic effects were observed on normal fresh orproliferating human PBMC (Li, Y., et al., Mol Med, 6 (2000) 1008).

β-Lapachone and its derivatives have also been synthesized and tested asanti-viral and anti-parasitic agents (Goncalves, A. M., et al. Mol.Biochem. Parasitology, I (1 980) 167-176; Schaffner-Sabba, K., et al., JMed, Chem., 27 (1984) 990-994).

β-Lapachone has been shown to be a DNA repair inhibitor that sensitizescells to DNA-damaging agents including radiation (Boothman, D. A. etal., Cancer Res, 47 (1987) 5361; Boorstein, R. J., et al., Biochem.Biophys. Commun., 117 (1983) 30). β-Lapachone has been asserted to havepotent in vitro inhibition of human DNA Topoisomerases I (Li, C. J., etal., J Biol. Chem., 268 (1993) 22463) and II (Frydruan, B. et al.,Cancer Res. 57 (1997) 620) with novel mechanisms of action.Topoisomerase I is an enzyme that unwinds the DNA that makes up thechromosomes. The chromosomes must be unwound in order for the cell touse the genetic information to synthesize proteins; β-lapachone may keepthe chromosomes wound tight, so that the cell cannot make proteins. As aresult, the cell stops growing. Because cancer cells are constantlyreplicating and circumvent many mechanisms that restrict replication innormal cells, they are more vulnerable to topoisomerase inhibition thanare normal cells.

Another possible intracellular target for β-lapachone in tumor cells isthe enzyme NAD(P)H:quinone oxidoreductase (NQO1, E.C. 1.6.99.2).β-lapachone is bioactivated by the NQO1 enzyme, which is a ubiquitousflavoprotein found in most eukaryotic cells. This enzyme catalyzes atwo-electron reduction of various quinones, utilizing either NADH orNADPH as electron donors. Biochemical studies suggest that reduction ofβ-lapachone by NQO1 leads to a “futile cycling” between the quinone andhydroquinone forms with a concomitant loss of reduced NADH or NAD(P)H(Pink, J. J. et al, J Biol Chem., 275 (2000) 5416). The exhaustion ofthese reduced enzyme cofactors may be a critical factor for theactivation of the apoptotic pathway after β-lapachone treatment. Thehuman NQO1 gene encodes a 30 kDa protein that is expressed in atissue-dependent manner. More importantly, NQO1 is over-expressed (up to20-fold) in a number of tumors, including breast, colon and lungcancers, compared with adjacent normal tissue (1-4). Over-expression ofNQO1 in cancerous cells makes it an ideal target for tumor-selectivedrug therapies with minimal toxicities to healthy cells.

Despite the potency and selectivity of β-lap in killing cancer cells invitro, the low water solubility of β-lapachone (0.04 mg/ml or 0.16 mM)limits its potential for systemic administration and clinicalapplications in vivo. β-lapachone is highly insoluble in water and hasonly limited solubility in common solvent systems used for topical andparenteral administration. As a result, there is a need for improvedformulations of β-lapachone for therapeutic purposes that are both safeand readily bioavailable to the subject to which the formulation isadministered.

SUMMARY OF THE INVENTION

In part, the present invention is directed to a system comprising alapachone or a prodrug thereof and a polymer, such as a biocompatibleand optionally biodegradable polymer, methods for treatment using thesubject polymer compositions, and methods of making and using the same.In another part, the present invention includes inclusion complexes of alapachone or a prodrug thereof and a cyclodextrin, preferably aβ-cyclodextrin, e.g., a hydroxyalkyl cyclodextrin such as hydroxypropylβ-cyclodextrin, e.g., to improve the solubility or bioavailability ofthe lapachone.

Lapachones are known to have activity against neoplastic cells, asdescribed in U.S. Pat. Nos. 5,969,163, 5,824,700, and 5,763,625.Antiviral activity (in combination with xanthine) or reversetranscriptase inhibitory activity for lapachones is suggested in U.S.Pat. Nos. 5,641,773 and 4,898,870, while antifungal and trypanosidalactivity of lapachones is suggested in U.S. Pat. Nos. 5,985,331 and5,912,241. Accordingly, it is contemplated that the subject compositionswill be useful as antimalarial, antifungal, antiparasitic, and/orantiviral therapeutics. Additional discussion of uses for lapachones canbe found in U.S. patent applications Nos. 2003064913, 2003036515,2003013677, and 20030169135, and U.S. Pat. No. 6,245,807.

Thus, in one embodiment, the invention provides a lapachone or a prodrugthereof, such as described herein, e.g., having a structure of Formula Ior II, complexed with a β-cyclodextrin, e.g., a hydroxyalkylcyclodextrin such as hydroxypropyl β-cyclodextrin. The complex,preferably an inclusion complex, may be combined with a pharmaceuticallyacceptable excipient to provide a pharmaceutical formulation, e.g.,suitable for administration to a patient, that may be useful as anantimalarial, antifungal, antiparasitic, and/or antiviral therapeutic.

In another aspect, the invention provides a drug delivery system,comprising a lapachone or a prodrug thereof as described herein, such asa lapachone of Formula I or II, incorporated in a biocompatible polymer.In certain embodiments, the lapachone is admixed with the polymer,although the lapachone may be coated with the polymer, or may otherwisebe placed in contact with the polymer. The system may be an implant,such as a millirod dimensioned to position two radiation seeds apredetermined distance apart, or may be microparticles and/ornanoparticles, such as microspheres and/or nanospheres. In certainembodiments, the lapachone is provided as an inclusion complex withhydroxypropyl cyclodextrin. The polymer may include one or more ofpoly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA),polyethylene glycol (PEG), polysebacic acid (PSA), or a polyanhydride,or copolymers of any of these. In certain embodiments, a diagnosticagent, an imaging agent, or an adjuvant is also incorporated in thepolymer.

In one aspect, the subject polymers may be biocompatible, biodegradableor both. The polymers comprise monomeric units arranged to form polymersas described in detail below. In the subject polymers, the chemicalstructure of certain of the monomeric units may be varied to achieve avariety of desirable physical or chemical characteristics, including,for example, release profiles, or handling characteristics of theresulting polymer composition.

In certain embodiments, one or more additional biologically activeagents may be encapsulated by the subject polymer. In certainembodiments, such agents may include compounds that counteract sideeffects induced by the lapachone, such as dicoumarol, while in otherembodiments, such agents may include compounds that enhance or augmentthe desired effects of the lapachone. In certain embodiments, thesubject polymers are combined with one or more other materials thatalter the physical/or and chemical properties of the resulting polymer,including, for example, the release profile of the resulting polymercomposition for an incorporated biologically active agent. Examples ofsuch materials include biocompatible plasticizers, delivery agents,fillers, and the like.

In certain embodiments, the subject compositions are in the form ofmicrospheres. In other embodiments, the subject compositions are in theform of nanospheres. In one embodiment, the microspheres or thenanospheres are formed in an emulsion. In another embodiment, thesubject compositions of the present invention may be lyophilized orsubjected to another appropriate drying technique such as spray dryingand subsequently used directly, e.g., inhaled or injected as powderusing an appropriate powder inhalation or injecting device, orrehydrated before use.

In another aspect, the present invention is directed to methods of usingthe subject polymer compositions for prophylactic or therapeutictreatment. In certain instances, the subject compositions may be used toprevent or treat a disease or condition in an animal, such as a human.In certain embodiments, use of the subject compositions that release ina sustained manner a therapeutic agent allow for different treatmentregimens than are possible with other modes of administration of suchtherapeutic agent.

In another aspect, the efficacy of treatment using the subjectcompositions may be compared to treatment regimens known in the art inwhich a therapeutic and/or biologically active agent is not encapsulatedwith a subject polymer, e.g., the agent is combined with a differentpolymer, or is administered substantially free of a polymer. Agents thatmay be encapsulated in the subject compositions include imaging anddiagnostic agents (such as radioopaque agents, labeled antibodies,labeled nucleic acid probes, dyes, such as colored or fluorescent dyes,etc.) and adjuvants (radiosensitizers, transfection-enhancing agents(such as chloroquine and analogs thereof), chemotactic agents andchemoattractants, peptides that modulate cell adhesion and/or cellmobility, cell permeabilizing agents, inhibitors of multidrug resistanceand/or efflux pumps, etc.).

In another aspect, the subject polymers may be used in the manufactureof a medicament for any number of uses including, for example, treatingany disease or other treatable condition of a patient. In still otheraspects, the present invention is directed to a method for formulatingpolymers and compositions of the present invention in a pharmaceuticallyacceptable carrier.

In other embodiments, this invention contemplates a kit includingsubject compositions, and optionally instructions for their use. Usesfor such kits include, for example, therapeutic applications. In certainembodiments, the subject compositions contained in any kit have beenlyophilized and/or spray dried and may require rehydration before use.For example, in one embodiment, the invention provides a kit comprisinga lapachone or a prodrug thereof as described herein, such as alapachone of Formula I or II, a β-cyclodextrin (optionally ahydroxyalkyl cyclodextrin, such as hydroxypropyl β-cyclodextrin, andinstructions for combining the lapachone and β-cyclodextrin to form acomplex and administering the complex to a patient

In certain embodiments, microparticles of the invention may be packed inan inhaler for pulmonary delivery, e.g., for delivery locally to thelungs or for systemic delivery through the lungs and/or nasal passages.

In another aspect, the invention provides a method of inhibitingproliferation of a cancerous cell in a patient by administering to thepatient a composition as described herein. In certain embodiments, thecell overexpresses NQO1. In certain embodiments, the cell is a lungcancer cell (such as a non-small cell lung cancer cell), a breast cancercell, or a prostate cancer cell. In certain embodiments, the compositionis delivered to the patient by inhalation of microspheres comprising alapachone or a prodrug thereof and a biocompatible polymer, e.g., totreat lung cancer or deliver the lapachone systemically. In certainembodiments, the cell is a prostate cancer cell and the system isdelivered to the patient by implanting radioactive seeds spaced apart byat least one polymeric millirod comprising a lapachone or a prodrugthereof and a biocompatible polymer.

These embodiments of the present invention, other embodiments, and theirfeatures and characteristics will be apparent from the description,drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic diagram of the solubility equilibriums of β-lap inaqueous solutions containing cyclodextrin (CD). K_(s) and K_(c) are theequilibrium constants for β-lap solubility and formation of inclusioncomplex, respectively. [CD.β-lap], [β-Lap] and [CD] are theconcentrations of CD.β-lap complex, free β-lap and free CD,respectively.

FIG. 2. Phase solubility diagrams of β-lap as a function of cyclodextrinconcentrations at 25° C. A) α-CD, β-CD and γ-CD. B) HPβ-CD.

FIG. 3. (A) Chemical structure of β-lap and general geometry of HPβ-CD;(B) GROESY spectra of HPβ-CD.β-lap inclusion complex in D₂O at 25° C.;(C) the ¹H NMR spectrum of HPβ-CD.β-lap inclusion complex. Theconcentrations of HPβ-CD and β-lap are 58.8 and 10.6 mM, respectively.

FIG. 4. ¹H NMR (600 MHz) spectra of β-lap ([β-lap]=0.123 mM) as afunction of β-CD concentrations in D₂O: (A) phenyl protons (Hd, He, Hfand Hg); (B) methyl and methylene protons (Ha, Hb and Hc).

FIG. 5. Nonlinear curve fitting of (A) chemical shift of Hd in β-lap([β-lap]=0.123 mM) as a function of HPβ-CD (▪) and β-CD (●)concentrations in D₂O; (B) Splitting of Hc as a function of β-CDconcentrations in D₂O.

FIG. 6. A) Emission spectra of β-lap (61 μM) at different excitationwavelengths ranging from 257 to 360 nm. B) Emission spectra of β-lap (18μM) in different HPβ-CD concentrations at 25° C. (λ_(ex)=330 nm).

FIG. 7. Viability of log-phase MCF-7 cells exposed to β-lap in DMSO,HPβ-CD or β-CD inclusion complexes, as well as β-CD and HPβ-CD vehiclesalone. For β-lap in DMSO and β-lap inclusion complexes, the bottomhorizontal axis denotes the β-lap concentrations. The top horizontalaxis denotes the cyclodextrin concentrations in β-lap inclusioncomplexes as well as for vehicles (β-CD and HPβ-CD) alone. Experimentswere performed at least two times in triplicate to provide the standarddeviation.

FIG. 8. Effect of varied doses of β-lap in HPβ-CD inclusion complex andHPβ-CD (control group injected with 5000 mg of HPβ-CD/kg) on thesurvival of C57Blk/6 mice. Animals were injected i.p. at day, 1, 3, 6,8, 10, 13, 15, 17, 20 and 22.

FIG. 9 shows responses of an implanted cell line to compositions of thepresent invention.

FIG. 10. NQO1-dependent apoptosis of A549 or CC-10 NSCLC cells by β-lap.Human A549 NSCLC cells were exposed to 8 μM β-lap, ±50 μM dicoumarol for4 h, drugs were removed, and cells monitored for: survival (A); orWestern blot analyses of PARP cleavage (B) as noted (Pink, J. J.,Wuerzberger-Davis, S., Tagliarino, C., Planchon, S. M., Yang, X.,Froelich, C. J., and Bootliman, D. A. Exp Cell Res, 255: 144-155, 2000;Wuerzberger, S. M., Pink, J. J., Planchon, S. M., Byers, K. L.,Bornmann, W. G., and Boothman, D. A. Cancer Res, 58: 1876-1885, 1998).Dicoumarol alone had no effect on cell growth (survival), but blockedβ-lap-induced apoptotic PARP cleavage (see 60 kDa fragment inβ-lap-treated cells, but not in cells treated with β-lap+dicoumarol).NQO1 levels remained unchanged and indicated equal loading (B). Similarresults were found with CC-10 tumor cells, where dicoumarol blockedβ-lap cytotoxicity (C).

FIG. 11. Sustained release of β-lap from PLGA microspheres in PBS at 37°C. The loading density of β-lap in microspheres was 2.0±0.07%. Thehorizontal line at 0.035 mg is the predicted amount of β-lap to achieve10 μM in a 3 cm diameter tumor.

FIG. 12. Sustained release of β-lap from PLGA millirods in PBS at 37° C.The loading density of β-lap in millirods was 10%. The horizontal lineat 0.035 mg represents the amount of β-lap to achieve a 10 μMconcentration in a tumor of 3 cm in diameter.

FIG. 13. Stacked UV-Vis spectra of β-lap released at different timepoints. The similar signature peaks in UV-Vis spectra (UV absorbance vs.Wavelength) suggest that the released β-lap maintains its structuralintegrity.

FIG. 14. NQO1 expression enhances β-lap, but decreases menadione,cytotoxicity. Left, NQO1-containing (LN-NQ C11-4, 10) and NQO1-deficient(LN-pcDNA3) LNCaP clones were treated with 4-h pulses of various dosesof β-lap, ±50 μM dicoumarol. Colony forming ability assays (CFAs) wereperformed three times, each in triplicate. Open symbols: β-lap alone;Closed symbols: β-lap+50 μM dicoumarol. Right, a representativeNQO1-transfected LNCaP clone (LN-NQ Cl 10) and one LNCaP vector aloneclone (LN-pcDNA3) were treated with 4-h pulses of various doses ofmenadione and CFA assays were determined (Pink, J. J., Planchon, S. M.,Tagliarino, C., Varnes, M. E., Siegel, D. and Boothman, D. A. (2000) JBiol Chem 275, 5416-5424).

FIG. 15. NQO1 expression is required for radiosensitization of LNCaPcells by β-lap. NQO1⁻LNCaP pcDNA3 vector alone and LNCaP NQO1⁺ clone 2(NQCl2) cells were exposed to various IR doses, followed by variousconcentrations (in μM, 4-h) of β-lap. Changes in colony forming ability.In A, pcDNA3 -vector alone cells were tested. In B, same as A, exceptthat NQO1⁺LNCaP cells were used.

FIG. 16. β-Lap shows significant antitumor activity against MDA-MB-468NQO1⁺ human xenografts. Athymic nude mice bearing 20 cm³ tumors weretreated with the indicated mg/mk doses of β-lap i.p. every other day,beginning at day 26. Each line represents an individual tumor volume.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The present invention relates to strategies for the delivery of alapachone or a prodrug thereof to a patient in need thereof, includingsustained release delivery, through a wide variety of routes, includingmicrospheres and nanospheres for injection or inhalation. The polymerscan be prepared using clinically approved monomers, including lacticacid (LA), glycolic acid (GA), sebacic acid (SA),1,3-bis(carboxyphenoxy)propane (CPP), and blocks ofpoly(lactic-co-glycolic acid) (PLGA) and/or poly(ethylene glycol) (PEG)of various molecular weights. By varying their composition, theproperties of drug-loaded particles made from these polymers can beoptimized. For example, surface properties can be tuned to improveaerosolization efficiency; phagocytic particle clearance in the deeplung can be inhibited by the presence of PEG in the polymer backbone(and ultimately on the particle surface); and continuous drug deliverykinetics can be achieved with control over total duration (hours toweeks). These properties provide a great deal of flexibility for thedelivery of a wide range of drugs.

In certain embodiments, biodegradable, biocompatible polymers maybe usedto deliver an encapsulated therapeutic agent in addition to thelapachone. Agents that may be encapsulated in the subject compositionsinclude imaging and diagnostic agents (such as radioopaque agents,labeled antibodies, labeled nucleic acid probes, dyes, such as coloredor fluorescent dyes, etc.) and adjuvants (radiosensitizers,transfection-enhancing agents (such as chloroquine and analogs thereof),chemotactic agents and chemoattractants, peptides that modulate celladhesion and/or cell mobility, cell permeabilizing agents, inhibitors ofmultidrug resistance and/or efflux pumps, etc.). Particular compoundsthat have been investigated in combination with lapachones includetaxanes (such as paclitaxel and docetaxel), thalidomide, xanthine, andangiogenesis inhibitors. Accordingly, the present invention contemplatescompositions that comprise such agents in addition to a lapachone.

The present invention also relates to methods of administering suchcompositions, e.g., as part of a treatment regimen, for example, byinhalation, by implantation, or by injection, e.g., subcutaneously,intramuscularly, or intravenously.

In certain embodiments, the subject pharmaceutical compositions, underbiological conditions, e.g., upon contact with body fluids includingblood, spinal fluid, lymph or the like, release the encapsulated drugover a sustained or extended period (as compared to the release from anisotonic saline solution). Such a system may result in prolongeddelivery (over, for example, 8 to 800 hours, preferably 24 to 480 ormore hours) of effective amounts (e.g., 0.0001 mg/kg/hour to 10mg/kg/hour) of the drug. This dosage form may be administered as isnecessary depending on the subject being treated, the severity of theaffliction, the judgment of the prescribing physician, and the like.

In certain embodiments, lapachones selectively target cancer cells orother cells that express or overexpress NAD(P)H:quinone oxidoreductase(NQO1). NQO1 is over-expressed in a number of tumors, including breast,colon, lung, and liver cancers, as compared with surrounding normaltissue (Marin, A., et al., (1997) Br. J. Cancer 76:923-929; Malkinson,A. M., et al., (1992) Cancer Res. 52:4752-4757; Belinsky, M., et al.,(1993) Cancer Metastasis Rev. 12:103-117; Joseph, P., et al., (1994)Oncol. Res. 6:525-532). Thus, the invention contemplates the treatmentand/or prevention of a cancer characterized by overexpression of NQO1.Furthermore, lapachones act as radiosensitizers in such cells, and thusthe invention further contemplates the administration of lapachones inconjunction with radiotherapy, e.g., for the treatment of cancer,whether pre- or post-operative.

2. Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples, and appendedclaims are collected here. These definitions should be read in light ofthe remainder of the disclosure and understood as by a person of skillin the art.

The term “access device” is an art-recognized term and includes anymedical device adapted for gaining or maintaining access to an anatomicarea. Such devices are familiar to artisans in the medical and surgicalfields. An access device may be a needle, a catheter, a cannula, atrocar, a tubing, a shunt, a drain, or an endoscope such as otoscope,nasopharyngoscope, bronrchoscope, or any other endoscope adapted for usein the head and neck area, or any other medical device suitable forentering or remaining positioned within the preselected anatomic area.

The terms “biocompatible polymer” and “biocompatibility” when used inrelation to polymers are art-recognized. For example, biocompatiblepolymers include polymers that are neither themselves toxic to the host(e.g., an animal or human), nor degrade (if the polymer degrades) at arate that produces monomeric or oligomeric subunits or other byproductsat toxic concentrations in the host. In certain embodiments of thepresent invention, biodegradation generally involves degradation of thepolymer in an organism, e.g., into its monomeric subunits, which may beknown to be effectively non-toxic. Intermediate oligomeric productsresulting from such degradation may have different toxicologicalproperties, however, or biodegradation may involve oxidation or otherbiochemical reactions that generate molecules other than monomericsubunits of the polymer. Consequently, in certain embodiments,toxicology of a biodegradable polymer intended for in vivo use, such asimplantation or injection into a patient, may be determined after one ormore toxicity analyses. It is not necessary that any subject compositionhave a purity of 100% to be deemed biocompatible. Hence, a subjectcomposition may comprise 99%, 98%, 97%, 96%, 95%, 90% 85%, 80%, 75% oreven less of biocompatible polymers, e.g., including polymers and othermaterials and excipients described herein, and still be biocompatible.

To determine whether a polymer or other material is biocompatible, itmay be necessary to conduct a toxicity analysis. Such assays are wellknown in the art. One example of such an assay may be performed withlive carcinoma cells, such as GT3TKB tumor cells, in the followingmanner: the sample is degraded in 1 M NaOH at 37° C. until completedegradation is observed.

The solution is then neutralized with 1 M HCl. About 200 μL of variousconcentrations of the degraded sample products are placed in 96-welltissue culture plates and seeded with human gastric carcinoma cells(GT3TKB) at 104/well density. The degraded sample products are incubatedwith the GT3TKB cells for 48 hours. The results of the assay may beplotted as % relative growth vs. concentration of degraded sample in thetissue-culture well. In addition, polymers and formulations of thepresent invention may also be evaluated by well-known in vivo tests,such as subcutaneous implantations in rats to confirm that they do notcause significant levels of irritation or inflammation at thesubcutaneous implantation sites.

The term “biodegradable” is art-recognized, and includes polymers,compositions and formulations, such as those described herein, that areintended to degrade during use. Biodegradable polymers typically differfrom non-biodegradable polymers in that the former may be degradedduring use. In certain embodiments, such use involves in vivo use, suchas in vivo therapy, and in other certain embodiments, such use involvesin vitro use. In general, degradation attributable to biodegradabilityinvolves the degradation of a biodegradable polymer into its componentsubunits, or digestion, e.g., by a biochemical process, of the polymerinto smaller, non-polymeric subunits. In certain embodiments, twodifferent types of biodegradation may generally be identified. Forexample, one type of biodegradation may involve cleavage of bonds(whether covalent or otherwise) in the polymer backbone. In suchbiodegradation, monomers and oligomers typically result, and even moretypically, such biodegradation occurs by cleavage of a bond connectingone or more of subunits of a polymer. In contrast, another type ofbiodegradation may involve cleavage of a bond (whether covalent orotherwise) internal to sidechain or that connects a side chain to thepolymer backbone. For example, a therapeutic agent or other chemicalmoiety attached as a side chain to the polymer backbone may be releasedby biodegradation. In certain embodiments, one or the other or bothgenerally types of biodegradation may occur during use of a polymer.

As used herein, the term “biodegradation” encompasses both general typesof biodegradation. The degradation rate of a biodegradable polymer oftendepends in part on a variety of factors, including the chemical identityof the linkage responsible for any degradation, the molecular weight,crystallinity, biostability, and degree of cross-linking of suchpolymer, the physical characteristics (e.g., shape and size) of theimplant, and the mode and location of administration. For example, thegreater the molecular weight, the higher the degree of crystallinity,and/or the greater the biostability, the biodegradation of anybiodegradable polymer is usually slower. The term “biodegradable” isintended to cover materials and processes also termed “bioerodible”.

In certain embodiments wherein the biodegradable polymer also has atherapeutic agent or other material associated with it, thebiodegradation rate of such polymer may be characterized by a releaserate of such materials. In such circumstances, the biodegradation ratemay depend on not only the chemical identity and physicalcharacteristics of the polymer, but also on the identity of material(s)incorporated therein.

In certain embodiments, polymeric formulations of the present inventionbiodegrade within a period that is acceptable in the desiredapplication. In certain embodiments, such as in vivo therapy, suchdegradation occurs in a period usually less than about five years, oneyear, six months, three months, one month, fifteen days, five days,three days, or even one day on exposure to a physiological solution witha pH between 6 and 8 having a temperature of between 25 and 37° C. Inother embodiments, the polymer degrades in a period of between about onehour and several weeks, depending on the desired application.

The term “drug delivery device” is an art-recognized term and refers toany medical device suitable for the application of a drug or therapeuticagent to a targeted organ or anatomic region. The term includes, withoutlimitation, those formulations of the compositions of the presentinvention that release the therapeutic agent into the surroundingtissues of an anatomic area. The term further includes those devicesthat transport or accomplish the instillation of the compositions of thepresent invention towards the targeted organ or anatomic area, even ifthe device itself is not formulated to include the composition. As anexample, a needle or a catheter through which the composition isinserted into an anatomic area or into a blood vessel or other structurerelated to the anatomic area is understood to be a drug delivery device.As a further example, a stent or a shunt or a catheter that has thecomposition included in its substance or coated on its surface isunderstood to be a drug delivery device.

When used with respect to a therapeutic agent or other material, theterm “sustained release” is art-recognized. For example, a subjectcomposition which releases a substance over time may exhibit sustainedrelease characteristics, in contrast to a bolus type administration inwhich the entire amount of the substance is made biologically availableat one time. For example, in particular embodiments, upon contact withbody fluids including blood, spinal fluid, lymph or the like, thepolymer matrices (formulated as provided herein and otherwise as knownto one of skill in the art) may undergo gradual degradation (e.g.,through hydrolysis) with concomitant release of any materialincorporated therein, e.g., an therapeutic and/or biologically activeagent, for a sustained or extended period (as compared to the releasefrom a bolus). This release may result in prolonged delivery oftherapeutically effective amounts of any incorporated therapeutic agent.Sustained release will vary in certain embodiments as described ingreater detail below.

The term “delivery agent” is an art-recognized term, and includesmolecules that facilitate the intracellular delivery of a therapeuticagent or other material. Examples of delivery agents include: sterols(e.g., cholesterol) and lipids (e.g., a cationic lipid, virosome orliposome).

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions t o reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

The term “microspheres” is art-recognized, and includes substantiallyspherical colloidal structures, e.g., formed from biocompatible polymerssuch as subject compositions, having a size ranging from about one orgreater up to about 1000 microns. In general, “microcapsules”, also anart-recognized term, may be distinguished from microspheres, becausemicrocapsules are generally covered by a substance of some type, such asa polymeric formulation. The term “microparticles” is art-recognized,and includes microspheres and microcapsules, as well as structures thatmay not be readily placed into either of the above two categories, allwith dimensions on average of less than about 1000 microns. If thestructures are less than about one micron in diameter, then thecorresponding art-recognized terms “nanosphere,” “nanocapsule,” and“nanoparticle” may be utilized. In certain embodiments, the nanospheres,nancapsules and nanoparticles have a size an average diameter of about500, 200, 100, 50 or 10 nm.

A composition comprising microspheres may include particles of a rangeof particle sizes. In certain embodiments, the particle sizedistribution may be uniform, e.g., within less than about a 20% standarddeviation of the median volume diameter, and in other embodiments, stillmore uniform or within about 10% of themedian volume diameter.

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and includewithout limitation intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The term “treating” is art-recognized and includes preventing a disease,disorder or condition from occurring in an animal which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it; inhibiting the disease, disorder orcondition, e.g., impeding its progress; and relieving the disease,disorder, or condition, e.g., causing regression of the disease,disorder and/or condition. Treating the disease or condition includesameliorating at least one symptom of the particular disease orcondition, even if the underlying pathophysiology is not affected, suchas treating the pain of a subject by administration of an analgesicagent even though such agent does not treat the cause of the pain.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “preventing” is art-recognized, and when used in relation to acondition; such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,solvent or encapsulating material involved in carrying or transportingany subject composition, from one organ, or portion of the body, toanother organ, or portion of the body. Each carrier must be “acceptable”in the sense of being compatible with the other ingredients of a subjectcomposition and not injurious to the patient. In certain embodiments, apharmaceutically acceptable carrier is non-pyrogenic. Some examples ofmaterials which may serve as pharmaceutically acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically acceptable salts” is art-recognized, andincludes relatively non-toxic, inorganic and organic acid addition saltsof compositions, including without limitation, analgesic agents,therapeutic agents, other materials and the like. Examples ofpharmaceutically acceptable salts include those derived from mineralacids, such as hydrochloric acid and sulfuric acid, and those derivedfrom organic acids, such as ethanesulfonic acid, benzenesulfonic acidD-toluenesulfonic acid, and the like. Examples of suitable inorganicbases for the formation of salts include the hydroxides, carbonates, andbicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium,aluminum, zinc and the like. Salts may also be formed with suitableorganic bases, including those that are non-toxic and strong enough toform such salts. For purposes of illustration, the class of such organicbases may include mono-, di-, and trialkylamines, such as methylamine,dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylaminessuch as mono-, di-, and triethanolamine; amino acids, such as arginineand lysine; guanidine; N-methylglucosamine; N-methylglucamine;L-glutamine; N-methylpiperazine; morpholine; ethylenediamine;N-benzylphenethylamine; (trihydroxymethyl)aminoethane; and the like.See, for example, J. Pharm. Sci. 66: 1-19 (1977).

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as primates, mammals,and vertebrates.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” areart-recognized, and include the administration of a subject composition,therapeutic or other material at a site remote from the disease beingtreated. Administration of an agent directly into, onto, or in thevicinity of a lesion of the disease being treated, even if the agent issubsequently distributed systemically, may be termed “local” or“topical” or “regional” administration, other than directly into thecentral nervous system, e.g., by subcutaneous administration, such thatit enters the patient's system and, thus, is subject to metabolism andother like processes.

The phrase “therapeutically effective amount” is an art-recognized term.In certain embodiments, the term refers to an amount of the therapeuticagent that, when incorporated into a polymer of the present invention,produces some desired effect at a reasonable benefit/risk ratioapplicable to any medical treatment. In certain embodiments, the termrefers to that amount necessary or sufficient to eliminate or reducesensations of pain for a period of time. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective. amount of aparticular compound without necessitating undue experimentation.

The term “ED₅₀” is art-recognized. In certain embodiments, ED₅₀ meansthe dose of a drug that produces 50% of its maximum response or effect,or, alternatively, the dose that produces a pre-determined response in50% of test subjects or preparations.

The term “LD₅₀” is art-recognized. In certain embodiments, LD₅₀ meansthe dose of a drug that is lethal in 50% of test subjects. The term“therapeutic index” is an art-recognized term that refers to thetherapeutic index of a drug, defined as LD₅₀/ED₅₀.

The terms “incorporated” and “encapsulated” are art-recognized when usedin reference to a therapeutic agent, or other material and a polymericcomposition, such as a composition of the present invention. In certainembodiments, these terms include incorporating, formulating, orotherwise including such agent into a composition that allows forrelease, such as sustained release, of such agent in the desiredapplication. The terms contemplate any manner by which a therapeuticagent or other material is incorporated into a polymer matrix, includingfor example: attached to a monomer of such polymer (by covalent, ionic,or other binding interaction), physical admixture, enveloping the agentin a coating layer of polymer, and having such monomer be part of thepolymerization to give a polymeric formulation, distributed throughoutthe polymeric matrix, appended to the surface of the polymeric matrix(by covalent or other binding interactions), encapsulated inside thepolymeric matrix, etc. The term “co-incorporation” or “co-encapsulation”refers to-the incorporation of a therapeutic agent or other material andat least one other therapeutic agent or other material in a subjectcomposition.

More specifically, the physical form in which any therapeutic agent orother material is encapsulated in polymers may vary with the particularembodiment. For example, a therapeutic agent or other material may befirst encapsulated in a microsphere and then combined with the polymerin such a way that at least a portion of the microsphere structure ismaintained. Alternatively, a therapeutic agent or other material may besufficiently immiscible in the polymer of the invention that it isdispersed as small droplets, rather than being dissolved, in thepolymer. Any form of encapsulation or incorporation is contemplated bythe present invention, in so much as the release, preferably sustainedrelease, of any encapsulated therapeutic agent or other materialdetermines whether the form of encapsulation is sufficiently acceptablefor any particular use.

The term “biocompatible plasticizer” is art-recognized, and includesmaterials which are soluble or dispersible in the compositions of thepresent invention, which increase the flexibility of the polymer matrix,and which, in the amounts employed, are biocompatible. Suitableplasticizers are well known in the art and include those disclosed inU.S. Pat. Nos. 2,784,127 and 4,444,933. Specific plasticizers include,by way of example, acetyl tri-n-butyl citrate (c. 20 weight percent orless), acetyltrihexyl citrate (c. 20 weight percent or less), butylbenzyl phthalate, dibutylphthalate, dioctylphthalate, n-butyryltri-n-hexyl citrate, diethylene glycol dibenzoate (c. 20 weight percentor less) and the like.

The terms ‘amine’ and ‘amino’ are art-recognized and refer to bothunsubstituted and substituted amines as well as ammonium salts, e.g., ascan be represented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent hydrogen or ahydrocarbon substituent, or R₉ and R₁₀ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure. In preferred embodiments, none of R₉, R₁₀, andR′₁₀ is acyl, e.g., R₉, R₁₀, and R′₁₀ are selected from hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, carbocyclic aliphatic, and heterocyclicaliphatic. The term ‘alkylamine’ as used herein means an amine group, asdefined above, having at least one substituted or unsubstituted alkylattached thereto. Amino groups that are positively charged (e.g., R′₁₀is present) are referred to as ‘ammonium’ groups. In amino groups otherthan ammonium groups, the amine is preferably basic, e.g., its conjugateacid has a pK_(a) above 7.

The terms ‘amido’ and ‘amide’ are art-recognized as an amino-substitutedcarbonyl, such as a moiety that can be represented by the generalformula:

wherein R₉ and R₁₀ are as defined above. In certain embodiments, theamide will include imides.

‘Alkyl’ refers to a saturated or unsaturated hydrocarbon chain having 1to 18 carbon atoms, preferably 1 to 12, more preferably 1 to 6, morepreferably still 1 to 4 carbon atoms. Alkyl chains may be straight(e.g., n-butyl) or branched (e.g., sec-butyl, isobutyl, or t-butyl).Preferred branched alkyls have one or two branches, preferably onebranch. Preferred alkyls are saturated. Unsaturated alkyls have one ormore double bonds and/or one or more triple bonds. Preferred unsaturatedalkyls have one or two double bonds or one triple bond, more preferablyone double bond. Alkyl chains may be unsubstituted or substituted withfrom 1 to 4 substituents. Preferred alkyls are unsubstituted. Preferredsubstituted alkyls are mono-, di-, or trisubstituted. Preferred alkylsubstituents include halo, haloalkyl, hydroxy, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl,and heteroaryl.

The terms ‘alkenyl’ and ‘alkynyl’ refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. When not otherwise indicated, the terms alkenyl andalkynyl preferably refer to lower alkenyl and lower alkynyl groups,respectively. When the term alkyl is present in a list with the termsalkenyl and alkynyl, the term alkyl refers to saturated alkyls exclusiveof alkenyls and alkynyls.

The terms ‘alkoxyl’ and ‘alkoxy’ as used herein refer to an —O-alkylgroup. Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, and the like. An ‘ether’ is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon an ether can be an alkoxyl, Or another moietysuch as —O-aryl, —O-heteroaryl, —O-heteroalkyl, —O-aralkyl,—O-heteroaralkyl, —O-carbocylic aliphatic, or —O-heterocyclic aliphatic.

The term ‘alkylthio’ refers to an —S-alkyl group. Representativealkylthio groups include methylthio, ethylthio, and the like.‘Thioether’ refers to a sulfur atom bound to two hydrocarbonsubstituents, e.g., an ether wherein the oxygen is replaced by sulfur.Thus, a thioether substituent on a carbon atom refers to ahydrocarbon-substituted sulfur atom substituent, such as alkylthio orarylthio, etc.

The term ‘aralkyl’, as used herein, refers to an alkyl group substitutedwith an aryl group.

‘Aryl ring’ refers to an aromatic hydrocarbon ring system. Aromaticrings are monocyclic or fused bicyclic ring systems, such as phenyl,naphthyl, etc. Monocyclic aromatic rings contain from about 5 to about10 carbon atoms, preferably from 5 to 7 carbon atoms, and mostpreferably from 5 to 6 carbon atoms in the ring. Bicyclic aromatic ringscontain from 8 to 12 carbon atoms, preferably 9 or 10 carbon atoms inthe ring. The term ‘aryl’ also includes bicyclic ring systems whereinonly one of the rings is aromatic, e.g:, the other ring is cycloalkyl,cycloalkenyl, or heterocyclyl. Aromatic rings may be unsubstituted orsubstituted with from 1 to about 5 substituents on the ring. Preferredaromatic ring substituents include: halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy, or any combination thereof.More preferred substituents include lower alkyl, cyano, halo, andhaloalkyl.

‘Carbocyclic aliphatic ring’ refers to a saturated or unsaturatedhydrocarbon ring. Carbocyclic aliphatic rings are not aromatic.Carbocyclic aliphatic rings are monocyclic, or are fused, Spiro, orbridged bicyclic ring systems. Mono cyclic carbocyclic aliphatic ringscontain from about 4 to about 10 carbon atoms, preferably from 4 to 7carbon atoms, and most preferably from 5 to 6 carbon atoms in the ring.Bicyclic carbocyclic aliphatic rings contain from 8 to 12 carbon atoms,preferably from 9 to 1 0 carbon atoms in the ring. Carbocyclic aliphaticrings may be unsubstituted or substituted with from 1 to 4 substituentson the ring. Preferred carbocyclic aliphatic ring substituents includehalo, cyano, alkyl, heteroalkyl, halo alkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo and haloalkyl. Preferred carbocyclic aliphatic rings include cyclopentyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. More preferredcarbocyclic aliphatic rings include cyclohexyl, cycloheptyl, andcyclooctyl.

The term ‘carbonyl’ is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, hydrocarbon substituent, or a pharmaceuticallyacceptable salt, R₁₁, represents a hydrogen or hydrocarbon substituent.Where X is an oxygen and R₁₁ or R₁₁, is not hydrogen, the formularepresents an ‘ester’. Where X is an oxygen, and R₁₁ is as definedabove, the moiety is referred to herein as a carboxyl group, andparticularly when R₁₁ is a hydrogen, the formula represents a‘carboxylic acid’. Where X is an oxygen, and R₁₁, is hydrogen, theformula represents a ‘formate’. In general, where the oxygen atom of theabove formula is replaced by sulfur, the formula represents a‘thiocarbonyl’ group. Where X is a sulfur and R₁₁ or R₁₁, is nothydrogen, the formula represents a ‘thioester.’ Where X is a sulfur andR₁₁ is hydrogen, the formula represents a ‘thiocarboxylic acid.’ Where Xis a sulfur and R₁₁, is hydrogen, the formula represents a‘thioformate.’ On the other hand, where X is a bond, R₁₁ is nothydrogen, and the carbonyl is bound to a hydrocarbon, the above formularepresents a ‘ketone’ group. Where X is a bond, R₁₁ is hydrogen, and thecarbonyl is bound to a hydrocarbon, the above formula represents an‘aldehyde’ or ‘formyl’ group.

‘Ci alkyl’ is an alkyl chain having i member atoms. For example, C4alkyls contain four carbon member atoms. C4 alkyls containing may besaturated or unsaturated with one or two double bonds (cis or trans) orone triple bond. Preferred C4 alkyls are saturated. Preferredunsaturated C4 alkyl have one double bond. C4 alkyl may be unsubstitutedor substituted with one or two substituents. Preferred substituentsinclude lower alkyl, lower heteroalkyl, cyano, halo, and haloalkyl.

‘Halogen’ refers to fluoro, chloro, bromo, or iodo substituents.Preferred halo are fluoro, chloro and bromo; more preferred are chloroand fluoro.

‘Haloalkyl’ refers to a straight, branched, or cyclic hydrocarbonsubstituted with one or more halo substituents. Preferred haloalkyl areC1-C12; more preferred are C1-C6; more preferred still are C1-C3.Preferred halo substituents are fluoro and chloro. The most preferredhaloalkyl is trifluoromethyl.

‘Heteroalkyl’ is a saturated or unsaturated chain of carbon atoms and atleast one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 1 to 18 member atoms (carbon andheteroatoms) in the chain, preferably 1 to 12, more preferably 1 to 6,more preferably still 1 to 4. Heteroalkyl chains may be straight orbranched. Preferred branched heteroalkyl have one or two branches,preferably one branch. Preferred heteroalkyl are saturated. Unsaturatedheteroalkyl have one or more double bonds and/or one or more triplebonds. Preferred unsaturated heteroalkyl have one or two double bonds orone triple bond, more preferably one double bond. Heteroalkyl chains maybe unsubstituted or substituted with from 1 to about 4 substituentsunless otherwise specified. Preferred heteroalkyl are unsubstituted.Preferred heteroalkyl substituents include halo, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,heteroaryl. For example, alkyl chains substituted with the followingsubstituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy,butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy,methoxyphenoxy, benzyloxy, alkoxycarbonylphenoxy, acyloxyphenoxy),acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy,carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di-C1-C3alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,carbamamido, ureido, guanidino).

‘Heteroatom’ refers to a multivalent non-carbon, atom, such as a boron,phosphorous, silicon, nitrogen, sulfur, or oxygen atom, preferably anitrogen, sulfur, or oxygen atom. Groups containing more than oneheteroatom may contain different heteroatoms.

‘Heteroaryl ring’ refers to an aromatic ring system containing carbonand from 1 to about 4 heteroatoms in the ring. Heteroaromatic rings aremonocyclic or fused bicyclic ring systems. Monocyclic heteroaromaticrings contain from about 5 to about 10 member atoms (carbon andheteroatoms), preferably from 5 to 7, and most preferably from 5 to 6 inthe ring. Bicyclic heteroaromatic rings contain from 8 to 12 memberatoms, preferably 9 or 10 member atoms in the ring. The term‘heteroaryl’ also includes bicyclic ring systems wherein only one of therings is aromatic e.g the other ring is cycloalkyl, cycloalkenyl, orheterocyclyl. Heteroaromatic rings may be unsubstituted or substitutedwith from 1 to about 4 substituents on the ring. Preferredheteroaromatic ring substituents include halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.Preferred heteroaromatic rings include thienyl, thiazolyl, oxazolyl,pyrrolyl, purinyl, pyrimidyl, pyridyl, and furanyl. More preferredheteroaromatic rings include thienyl, furanyl, and pyridyl.

‘Heterocyclic aliphatic ring’ is a non-aromatic saturated or unsaturatedring containing carbon and from 1 to about 4 heteroatoms in the ring,wherein no two heteroatoms are adjacent in the ring and preferably nocarbon in the ring attached to a heteroatom also has a hydroxyl, amino,or thiol group attached to it. Heterocyclic aliphatic rings aremonocyclic, or are fused or bridged bicyclic ring systems. Monocyclicheterocyclic aliphatic rings contain from about 4 to about 10 memberatoms (carbon and heteroatoms), preferably from 4 to 7, and mostpreferably from 5 to 6 member atoms in the ring. Bicyclic heterocyclicaliphatic rings contain from 8 to 12 member atoms, preferably 9 or 10member atoms in the ring. Heterocyclic aliphatic rings may beunsubstituted or substituted with from 1 to about 4 substituents on thering. Preferred heterocyclic aliphatic ring substituents include halo,cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, hydantoin,oxazoline, imidazolinetrione, triazolinone, quinoline, phthalazine,naphthyridine, quinoxaline, quinazoline, quinoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,phenazine, phenarsazine, phenothiazine, furazan, phenoxazine,pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine,morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like. Preferred heterocyclic aliphatic ringsinclude piperazyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl andpiperidyl. Heterocycles can also be polycycles.

The term ‘hydroxyl’ means —OH.

‘Lower alkyl’ refers to an alkyl chain comprised of 1 to 4, preferably 1to 3 carbon member atoms, more preferably 1 or 2 carbon member atoms.Lower alkyls may be saturated or unsaturated. Preferred lower alkyls aresaturated. Lower alkyls may be unsubstituted or substituted with one orabout two substituents. Preferred substituents on lower alkyl includecyano, halo, trifluoromethyl, amino, and hydroxyl. Throughout theapplication, preferred alkyl groups are lower alkyls. In preferredembodiments, a substituent designated herein as alkyl is a lower alkyl.Likewise, ‘lower alkenyl’ and ‘lower alkynyl’ have similar chainlengths.

‘Lower heteroalkyl’ refers to a heteroalkyl chain comprised of 1 to 4,preferably 1 to 3 member atoms, more preferably 1 to 2 member atoms.Lower heteroalkyl contain one or two non-adjacent heteroatom memberatoms. Preferred lower heteroalkyl contain one heteroatom member atom.Lower heteroalkyl may be saturated or unsaturated. Preferred lowerheteroalkyl are saturated. Lower heteroalkyl may be unsubstituted orsubstituted with one or about two substituents. Preferred substituentson lower heteroalkyl include cyano, halo, trifluoromethyl, and hydroxyl.

‘Mi heteroalkyl’ is a heteroalkyl chain having i member atoms. Forexample, M4 heteroalkyls contain one or two non-adjacent heteroatommember atoms. M4 heteroalkyls containing 1 heteroatom member atom may besaturated or unsaturated with one double bond (cis or trans) or onetriple bond. Preferred M4 heteroalkyl containing 2 heteroatom memberatoms are saturated. Preferred unsaturated M4 heteroalkyl have onedouble bond. M4 heteroalkyl maybe unsubstituted or substituted with oneor two substituents. Preferred substituents include lower alkyl, lowerheteroalkyl, cyano, halo, and haloalkyl.

‘Member atom’ refers to a polyvalent atom (e.g., C, O, N, or S atom) ina chain or ring system that constitutes a part of the chain or ring. Forexample, in cresol, six carbon atoms are member atoms of the ring andthe oxygen atom and the carbon atom of the methyl substituent are notmember atoms of the ring.

‘Pharmaceutically acceptable salt’ refers to a cationic salt formed atany acidic (e.g., hydroxamic or carboxylic acid) group, or an anionicsalt formed at any basic (e.g., amino or guanidino) group. Such saltsare well known in the art. See e.g., PCT Publication 87/05297, Johnstonet al., published Sep. 11, 1987, incorporated herein by reference. Suchsalts are made by methods known to one of ordinary skill in the art. Itis recognized that the skilled artisan may prefer one salt over anotherfor improved solubility, stability, formulation ease, price and thelike. Determination and optimization of such salts is within the purviewof the skilled artisan's practice. Preferred cations include the alkalimetals (such as sodium and potassium), and alkaline earth metals (suchas magnesium and calcium) and organic cations, such astrimethylammonium, tetrabutylammonium, etc. Preferred anions includehalides (such as chloride), sulfonates, carboxylates, phosphates, andthe like. Clearly contemplated in such salts are addition salts that mayprovide an optical center where once there was none. For example, achiral tartrate salt may be prepared from the compounds of theinvention. This definition includes such chiral salts.

‘Phenyl’ is a six-membered monocyclic aromatic ring that may or may notbe substituted with from 1 to 5 substituents. The substituents may belocated at the ortho, meta or para position on the phenyl ring, or anycombination thereof. Preferred phenyl substituents include: halo, cyano,lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combinationthereof. More preferred substituents on the phenyl ring include halo andhalo alkyl. The most preferred substituent is halo.

The term ‘sulfhydryl’ means —SH, and the term ‘sulfonyl’ means —SO₂—.

A ‘substitution’ or ‘substituent’ on a small organic molecule generallyrefers to a position on a multi-valent atom bound to a moiety other thanhydrogen, e.g., a position on a chain or ring exclusive of the memberatoms of the chain or ring. Such moieties include those defined hereinand others as are known in the art, for example, halogen, alkyl,alkenyl, alkynyl, azide, haloalkyl, hydroxyl, carbonyl (such ascarboxyl, alkoxycarbonyl, formyl, ketone, or acyl), thiocarbonyl (suchas thioester, thioacetate, or thioformate), alkoxyl, phosphoryl,phosphonate, phosphinate, amine, amide, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, silyl, ether, cycloalkyl, heterocyclyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, heteroaralkyl, aralkyl,aryl or heteroaryl. It will be understood by those skilled in the artthat certain substituents, such as aryl, heteroaryl, polycyclyl, alkoxy,alkylamino, alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, can themselves besubstituted, if appropriate. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that ‘substitution’ or ‘substitutedwith’ includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, hydrolysis, etc.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The abbreviations Me. Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, and methane sulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The phrase ‘protecting group’ as used herein means temporarysubstituents that protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991; and Kocienski, P. J. Protecting Groups, Georg Thieme Verlag: NewYork, 1994).

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term ‘hydrocarbon’ is contemplatedto include all permissible compounds or moieties having at least onecarbon-hydrogen bond. In a broad aspect, the permissible hydrocarbonsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Contemplated equivalents of the compounds described herein includecompounds which otherwise correspond thereto, and which have the sameuseful properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants that are in themselves known, but are not mentioned here.

3. Lapachones and Methods of Preparing Them

The present invention contemplates the delivery of β-lapachone and/orderivatives or analogs thereof, collectively referred to herein aslapachones. A wide variety of lapachone analogs which retain apharmacologically important quinone moiety have been described. See, forexample, U.S. Pat. Nos. 6,245,807, 5,763,625, 5,824,700, 5,969,163, and5,977,187, PCT publications WO 94/04145. and WO 00/61142, as well as.Sabba. et. al., J Med Chem. 27:990-994 (1984); Molina Portela andStoppani, Biochem Pharm 51:275-283 (1996); and Goncalves et al.,Molecular and Biochemical Parasitology 1:1 67-176 (1998). Strategies forpreparing various lapachones are described in U.S. Pat. Nos. 6,458,974,5,969,163, and 5,763,625.

In certain embodiments, a lapachone has a structure of either Formula Ior Formula II:

-   -   wherein R and R₁ each independently represent H, hydroxy, amino,        amido, sulfhydryl, halogen, or substituted or unsubstituted        alkyl, alkenyl, heteroalkyl, carbocyclic aliphatic, carbocyclic        aliphatic alkyl, aryl, aralkyl, heterocyclic aliphatic,        heterocyclic aliphatic alkyl, heteroaryl, heteroaralkyl, or        alkoxy,    -   or a pharmaceutically acceptable salt thereof.

Alkyl groups preferably have from 1 to about 15 carbon atoms, morepreferably from 1 to about 10 carbon atoms, still more preferably from 1to about 6 carbon atoms. Alkenyl groups preferably have from 2 to 15carbon atoms, more preferably from 2 to about 10 carbon atoms, stillmore preferably from 2 to about 6 carbon atoms. Especially preferredalkenyl groups have 3 carbon atoms (i.e., 1-propenyl or 2-propenyl),with the allyl moiety being particularly preferred. Phenyl and naphthylare generally preferred aryl groups. Alkoxy groups include those alkoxygroups having one or more oxygen linkage and preferably have from 1 to15 carbon atoms, more preferably from 1 to about 6 carbon atoms.Substituted R and R₁ groups may be substituted at one or more availablepositions by one or more suitable groups such as, for example, alkylgroups such as alkyl groups having from 1 to 10 carbon atoms or from 1to 6 carbon atoms, alkenyl groups such as alkenyl groups having from 2to 10 carbon atoms or 2 to 6 carbon atoms, aryl groups having from 6 to10 carbon atoms, halogen such as fluoro, chloro, and bromo, and N, O, orS, including heteroalkyl, e.g., heteroalkyl having one or more of saidhetero atom linkages (and thus including alkoxy, aminoalkyl andthioalkyl) and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.

In certain embodiments, the lapachone is provided as an inclusioncomplex with a cyclodextrin, preferably a β-cyclodextrin, such ashydroxypropyl β-cyclodextrin, e.g., to improve the solubility of thelapachone. Such complexes maybe administered in admixture with apolymer, or in non-polymeric formulations, such as injectable solutionsand oral formulations.

In certain embodiments, the lapachone is provided as a prodrug. Althoughmany strategies for preparing prodrugs are widely known in the art, oneparticular method of forming prodrugs of lapachone involves theformation of a Schiff base by condensing the lapachone with a primaryamine, such as a substituted or unsubstituted alkyl, carbocyclicaliphatic, carbocyclic aliphatic alkyl, aryl, aralkyl, heterocyclicaliphatic, heterocyclic aliphatic alkyl, heteroaryl, or heteroaralkylamine. The resulting prodrug may thus have a structure of Formula III:

-   -   wherein R and R₁ are as defined above, and R′ represents a        substituted or unsubstituted alkyl, carbocyclic aliphatic,        carbocyclic aliphatic alkyl, aryl, aralkyl, heterocyclic        aliphatic, heterocyclic aliphatic alkyl, heteroaryl, or        heteroaralkyl substituent. In certain embodiments, R′ is an        alkyl or aryl group. In embodiments wherein R′ represents        phenyl, the phenyl ring is optionally substituted, e.g., with a        nitro, methyl, methoxy, or halogen substituent. Varying the        substituent of the phenyl ring may affect the rate of hydrolysis        of the Schiff base, and thereby affect the rate at which the        prodrug is rendered active in a physiological environment.        4. Polymer Vehicles

A variety of polymers can be used in the preparation of lapachoneformulations. In certain embodiments, the polymer is biocompatible andbiodegradable, while in other embodiments, the polymer is merelybiocompatible.Suitable polymers include polypropylene, polyester,polyethylene vinyl acetate (PVA or EVA), polysebacic acid (PSA)polyethylene oxide (PEO; =poly(ethylene glycol), PEG), polypropyleneoxide, polycarboxylic acids, polyalkylacrylates, cellulose ethers,silicone, poly(dl-lactide-co-glycolide) (PLGA), various Eudragits (forexample, NE30D, RS PO and RL PO), polyalkyl-alkyacrylate copolymers,polyester-polyurethane block copolymers polyether-polyurethane blockcopolymers, polydioxanone, poly-(β-hydroxybutyrate), polylactic acid(PLA), polycaprolactone, polyglycolic acid (PGA), and copolymersthereof, including PEG-PLA, PEG-PSA, or PEG-PLGA copolymers. Certainsuch copolymers are discussed in detail in PCT publication WO 03/00237.

In certain preferred embodiments wherein the polymer is a copolymer ofPEO and another polymer, such as PSA, PLA, or PLGA, the ratio of PEG toits comonomer is between 5:50 and 5:120, preferably between about 5:70and about 5:100.

In certain embodiments, the polymeric chains of the subject compositionshave molecular weights (M_(w)) ranging from about 2000 or less to about300,000, 600,000 or 1,000,000 or more daltons, or alternatively at leastabout 10,000, 20,000, 30,000, 40,000, or 50,000 daltons, moreparticularly at least about 100,000 daltons. Number-average molecularweight (M_(n)) may also vary widely, but generally fall in the range ofabout 1,000 to about 200,000 daltons, preferably from about 10,000 toabout 100,000 daltons and, even more preferably, from about 8,000 toabout 50,000 daltons. Most preferably, M_(n) varies between about 12,000and 45,000 daltons. Within a given sample of a subject polymer, a widerange of molecular weights may be present. For example, molecules withinthe sample may have molecular weights that differ by a factor of 2, 5,10, 20, 50, 100, or more, or that differ from the average molecularweight by a factor of 2, 5, 10, 20, 50, 100, or more.

One method to determine molecular weight is by gel permeationchromatography (“GPC”), e.g., mixed bed columns, CH₂Cl₂ solvent, lightscattering detector, and off-line dn/dc. Other methods are known in theart.

In other embodiments, the polymer composition of the invention may be aflexible or flowable material. When the polymer used is itself flowable,the polymer composition of the invention, even when viscous, need notinclude a biocompatible solvent to be flowable, although trace orresidual amounts of biocompatible solvents may still be present.

While it is possible that the biodegradable polymer or the lapachone orother biologically active agent may be dissolved in a small quantity ofa solvent that is non-toxic to more efficiently produce an amorphous,monolithic distribution or a fine dispersion of the biologically activeagent in the flexible or flowable composition, it is an advantage of theinvention that, in a preferred embodiment, no solvent is needed to forma flowable composition. Moreover, the use of solvents is preferablyavoided, because once a polymer composition containing solvent is placedtotally or partially within the body, the solvent dissipates or diffusesaway from the polymer and must be processed and eliminated by the body,placing an extra burden on the body's clearance ability at a time whenthe illness (and/or other treatments for the illness) may have alreadydeleteriously affected it.

However, when a solvent is used to facilitate mixing or to maintain theflowability of the polymer composition of the invention, it should benon-toxic, otherwise biocompatible, and should be used in relativelysmall amounts. Solvents that are toxic should not be used in anymaterial to be placed even partially within a living body. Such asolvent also must not cause substantial tissue irritation or necrosis atthe site of administration.

Examples of suitable biocompatible solvents, when used, includeN-methyl-2-pyrrolidone, 2-pyrrolidone, ethanol, propylene glycol,acetone, methyl acetate, ethyl acetate, methyl ethyl ketone,dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, caprolactam,oleic acid, or 1-dodecylazacycoheptanone. Preferred solvents includeN-methyl pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, and acetonebecause of their solvating ability and their biocompatibility.

In certain embodiments, the subject polymers are soluble in one or morecommon organic solvents for ease of fabrication and processing. Commonorganic solvents include such solvents as chloroform, dichloromethane,dichloroethane, 2-butanone, butyl acetate, ethyl butyrate, acetone,ethyl acetate, dimethylacetamide, N-methyl pyrrolidone,dimethylformamide, and dimethylsulfoxide.

5. Applications

A. Therapeutic Compositions

In part, a biocompatible polymer composition of the present inventionincludes a biocompatible and optionally biodegradable polymer, such asone having the recurring monomeric units shown in one of the foregoingformulas, optionally including any other biocompatible and optionallybiodegradable polymer mentioned above or known in the art.

In addition to a lapachone or a prodrug thereof, the subjectcompositions may contain a “drug”, “therapeutic agent,” “medicament,” or“bioactive substance,” which are biologically, physiologically, orpharmacologically active substances that act locally or systemically inthe human or animal body. For example,a subject composition may includeany of the other compounds discussed above.

Various forms of the medicaments or biologically active materials may beused which are capable of being released from the polymer matrix intoadjacent tissues or fluids. They may be acidic, basic, or salts. Theymay be neutral molecules, polar molecules, or molecular complexescapable of hydrogen bonding. They may be in the form of ethers, esters,amides and the like, including prodrugs which are biologically activatedwhen injected into the human or animal body, e.g., by cleavage of anester or amide. An analgesic agent is also an example of a “bioactivesubstance.” Any additional bioactive substance in a subject compositionmay vary widely with the purpose for the composition. The term bioactiveagent includes without limitation, medicaments; vitamins; mineralsupplements; substances used for the treatment, prevention, diagnosis,cure or mitigation of disease or illness; or substances which affect thestructure or function of the body; or pro-drugs, which becomebiologically active or more active after they have been placed in apredetermined physiological environment.

Plasticizers and stabilizing agents known in the art may be incorporatedin polymers of the present invention. In certain embodiments, additivessuch as plasticizers and stabilizing agents are selected for theirbiocompatibility. In certain embodiments, the additives are lungsurfactants, such as 1,2-dipalmitoylphosphatidycholine (DPPC) andL-α-phosphatidylcholine (CC).

A composition of this invention may further contain one or more adjuvantsubstances, such as fillers, thickening agents or the like. In otherembodiments, materials that serve as adjuvants may be associated withthe polymer matrix. Such additional materials may affect thecharacteristics of the polymer matrix that results.

For example, fillers, such as bovine serum albumin (BSA) or mouse serumalbumin (MSA), may be associated with the polymer matrix. In certainembodiments, the amount of filler may range from about 0.1 to about 50%or more by weight of the polymer matrix, or about 2.5, 5, 10, 25, or 40percent. Incorporation of such fillers may affect the biodegradation ofthe polymeric material and/or the sustained release rate of anyencapsulated substance. Other fillers known to those of skill in theart, such as carbohydrates, sugars, starches, saccharides, cellulosesand polysaccharides, including mannitose and sucrose, may be used incertain embodiments in the present invention.

In other embodiments, spheronization enhancers facilitate the productionof subject polymeric matrices that are generally spherical in shape.Substances such as zein, microcrystalline cellulose or microcrystallinecellulose co-processed with sodium carboxymethyl cellulose may conferplasticity to the subject compositions as well as implant strength andintegrity. In particular embodiments, during spheronization, extrudatesthat are rigid, but not plastic, result in the formation of dumbbellshaped implants and/or a high proportion of fines, and extrudates thatare plastic, but not rigid, tend to agglomerate and form excessivelylarge implants. In such embodiments, a balance between rigidity andplasticity is desirable. The percent of spheronization enhancer in aformulation typically range from 10 to 90% (w/w).

In certain embodiments, a subject composition includes an excipient. Aparticular excipient may be selected based on its melting point,solubility in a selected solvent (e.g., a solvent that dissolves thepolymer and/or the therapeutic agent), and the resulting characteristicsof the microparticles.

Excipients may comprise a few percent, about 5%, 10%, 15%, 20%, 25%,30%, 40%, 50%, or higher percentage of the subject compositions.

Buffers, acids and bases may be incorporated in the subject compositionsto adjust their pH. Agents to increase the diffusion distance of agentsreleased from the polymer matrix may also be included.

Disintegrants are substances that, in the presence of liquid, promotethe disruption of the subject compositions. Disintegrants are most oftenused in implants, in which the function of the disintegrant is tocounteract or neutralize the effect of any binding materials used in thesubject formulation. In general, the mechanism of disintegrationinvolves moisture absorption and swelling by an insoluble material.

Examples of disintegrants include croscarmellose sodium and crospovidonewhich, in certain embodiments, may be incorporated into the polymericmatrices in the range of about 1-20% of total matrix weight. In othercases, soluble fillers such as sugars (mannitol and lactose) may also beadded to facilitate disintegration of implants.

Other materials may be used to advantage to control the desired releaserate of a therapeutic agent for a particular treatment protocol. Forexample, if the sustained release is too slow for a particularapplication, a pore-forming agent maybe added to generate additionalpores in the matrix. Any biocompatible water-soluble material may beused as the pore-forming agent. They may be capable of dissolving,diffusing or dispersing out of the formed polymer system whereupon poresand microporous channels are generated in the system. The amount ofpore-forming agent (and size of dispersed particles of such pore-formingagent, if appropriate) within the composition should affect the size andnumber of the pores in the polymer system.

Pore-forming agents include any pharmaceutically acceptable organic orinorganic substance that is substantially miscible in water and. bodyfluids and will dissipate from the forming and formed matrix intoaqueous medium or body fluids or water-immiscible substances thatrapidly degrade to water-soluble substances.

Suitable pore-forming agents include, for example, sugars such assucrose and dextrose, salts such as sodium chloride and sodiumcarbonate, and polymers such as hydroxylpropylcellulose,carboxymethylcellulose, polyethylene glycol, and PVP. The size andextent of the pores may be varied over a wide range by changing themolecular weight and. percentage of pore-forming agent incorporated intothe polymer system.

The charge, lipophilicity or hydrophilicity of any subject polymericmatrix may be modified by attaching in some fashion an appropriatecompound to the surface of the matrix. For example, surfactants may beused to enhance wettability of poorly soluble or hydrophobiccompositions. Examples of suitable surfactants include dextran,polysorbates and sodium lauryl sulfate. In general, surfactants are usedin low concentrations, generally less than about 5%.

Binders are adhesive materials that may be incorporated in polymericformulations to bind and maintain matrix integrity. Binders may be addedas dry powder or as solution. Sugars and natural and synthetic polymersmay act as binders.

Materials added specifically as binders are generally included in therange of about 0.5%-15% w/w of the matrix formulation. Certainmaterials, such as microcrystalline cellulose, also used as aspheronization enhancer, also have additional binding properties.

Various coatings may be applied to modify the properties of thematrices.

Three exemplary types of coatings are seal, gloss and enteric coatings.Other types of coatings having various dissolution or erosion propertiesmay be used to further modify subject matrices behavior, and suchcoatings are readily known to one of ordinary skill in the art.

The seal coat may prevent excess moisture uptake by the matrices duringthe application of aqueous based enteric coatings. The gloss coatgenerally improves the handling of the finished matrices. Water-solublematerials such as hydroxypropylcellulose may be used to seal coat andgloss coat implants. The seal coat and gloss coat are generally sprayedonto the matrices until an increase in weight between about 0.5% andabout 5%, often about 1% for a seal coat and about 3% for a gloss coat,has been obtained.

Enteric coatings consist of polymers which are insoluble in the low pH(less than 3.0) of the stomach, but are soluble in the elevated pH(greater than 4.0) of the small intestine. Polymers such as EUDRAGIT,RohmTech, Inc., Malden, Mass., and AQUATERIC, FMC Corp., Philadelphia,Pa., may be used and are layered as thin membranes onto the implantsfrom aqueous solution or suspension or by a spray drying method. Theenteric coat is generally sprayed to a weight increase of about one toabout 30%, preferably about 10 to about 15% and may contain coatingadjuvants such as plasticizers, surfactants, separating agents thatreduce the tackiness of the implants during coating, and coatingpermeability adjusters.

The present compositions may additionally contain one or more optionaladditives such as fibrous reinforcement, colorants, perfumes, rubbermodifiers, modifying agents, etc. In practice, each of these optionaladditives should be compatible with the resulting polymer and itsintended use. Examples of suitable fibrous reinforcement include PGAmicrofibrils, collagen microfibrils, cellulosic microfibrils, andolefinic microfibrils. The amount of each of these optional additivesemployed in the composition is an amount necessary to achieve thedesired effect.

B. Physical Structures of the Subject Compositions

The subject polymers may be formed in a variety of shapes. For example,in certain embodiments, subject polymer matrices may be presented in theform of microparticles or nanoparticles. Microspheres typically comprisea biodegradable polymer matrix incorporating a drug. Microspheres can beformed by a wide variety of techniques known to those of skill in theart. Examples of microsphere forming techniques include, but are notlimited to, (a) phase separation by emulsification and subsequentorganic solvent evaporation (including complex emulsion methods such asoil in water emulsions, water in oil emulsions and water-oil-wateremulsions); (b) coacervation-phase separation; (c) melt dispersion; (d)interfacial deposition; (e) in situ polymerization; (f) spray drying andspray congealing; (g) air suspension coating; and (h) pan and spraycoating. These methods, as well as properties and characteristics ofmicrospheres are disclosed in, for example, U.S. Pat. No. 4,652,441;U.S. Pat. No. 5,100,669; U.S. Pat. No. 4,526,938; WO 93/24150; EPA0258780 A2; U.S. Pat. No. 4,438,253; and U.S. Pat. 5,330,768, the entiredisclosures of which are incorporated by reference herein.

To prepare microspheres of the present invention, several methods can beemployed depending upon the desired application of the deliveryvehicles. Suitable methods include, but are not limited to, spraydrying, freeze drying, air drying, vacuum drying, fluidized-bed drying,milling, co-precipitation and critical fluid extraction. In the case ofspray drying, freeze drying, air drying, vacuum drying, fluidized-beddrying and critical fluid extraction; the components (stabilizingpolyol, bioactive material, buffers, etc.) are first dissolved orsuspended in aqueous conditions. In the case of milling, the componentsare mixed in the dried form and milled by any method known in the art.In the case of co-precipitation, the components are mixed in organicconditions and processed as described below. Spray drying can be used toload the stabilizing polyol with the bioactive material. The componentsare mixed under aqueous conditions and dried using precision nozzles toproduce extremely uniform droplets in a drying chamber. Suitable spraydrying machines include, but are not limited to, Buchi, NIRO, APV andLab-plant spray driers used according to the manufacturer'sinstructions.

The shape of microparticles and nanoparticles may be determined byscanning electron microscopy. Spherically shaped nanoparticles are usedin certain embodiments for circulation through the bloodstream. Ifdesired, the particles may be fabricated using known techniques intoother shapes that are more useful for a specific application.

In addition to intracellular delivery of a therapeutic agent, it alsopossible that particles of the subject compositions, such asmicroparticles or nanoparticles, may undergo endocytosis, therebyobtaining access to the cell. The frequency of such, an endocytosisprocess will likely depend on the size of any particle.

Microparticles may be administered by inhalation of a suitablecomposition using a suitable delivery device, such as an inhaler. Thismode of administration may be used for local delivery to the lung and/orsystemic delivery to the patient's bloodstream. Local administration ofa lapachgne in this fashion may be useful in the treatment or control oflung cancers, such a non-small cell lung cancer.

In other embodiments, polymeric formulations of lapachone are shaped asimplantable drug delivery devices to delivery therapeutic agents to alocalized tissue volume or mass. In certain embodiments, such devicesare useful for treating a tumor, such as breast cancer, ovarian cancer,or prostate cancer. In certain exemplary embodiments, drug deliverydevices are implanted into the breast, ovary, or prostate, or into thetissues immediately adjacent to the breast or prostate. The drugdelivery devices, detailed below, release the lapachone and optionallyadditional therapeutic agents or drugs over time to treat the cancer,and/or symptoms associated with the cancer, or symptoms associated withother treatment modalities for the cancer. For example, dicoumarol canbe incorporated in the implant(s) to alleviate some of the unwanted sideeffects induced by the lapachone.

According to one aspect of the invention, the polymeric implants arefashioned as spacers between brachytherapy seeds. Such spacers may, forexample, be millirods, e.g., about 0.6 to about 1.0 mm in diameter,about 3-7 mm in length, preferably adapted to fit through a 19-gaugeneedle. Brachytherapy treatments can generate pain, edema, andassociated voiding problems. In some embodiments, the brachytherapy drugdelivery spacer includes a biologically active agent that decreases, andpreferably eliminates, pain, swelling, and/or voiding symptoms followingbrachytherapy, and may also enhance (or be enhanced by) radiationtherapy. Certain known therapeutic compounds, such as 5FU andtriamcinolone acetonide, have beneficial effects in the treatment ofthese symptoms, especially in conjunction with brachytherapy.

Spacers may be placed between the radioactive seeds in the deliveryneedles of the brachytherapy machine, to keep the radioactive seeds intheir proper predetermined positions. The simultaneous use ofantiinflammatory agents that have a controlled and prolonged releaserate also limits the brachytherapy side effects, while the localizeddelivery of the agents to the prostate does not impose thechemotherapeutic load on the patient's entire system that is asignificant shortcoming of some prior systemic administration protocols.

In certain embodiments, solid articles useful in defining shape andproviding rigidity and structural strength to the polymeric matrices maybe used. For example, a polymer may be formed on a mesh or other weavefor implantation. A polymer may also be fabricated as a stent or as ashunt, adapted for holding open areas within body tissues or fordraining fluid from one body cavity or body lumen into another. Further,a polymer may be fabricated as a drain or a tube suitable for removingfluid from a post-operative site, and in some embodiments adaptable foruse with closed section drainage systems such as Jackson-Pratt drainsand the like as are familiar in the art.

The mechanical properties of the polymer may be important for theprocessability of making molded or pressed articles for implantation.For example, the glass transition temperature may vary widely but mustbe sufficiently lower than the temperature of decomposition toaccommodate conventional fabrication techniques, such, as compressionmolding, extrusion, or injection molding.

C. Biodegradability and Release Characteristics

In certain embodiments, the formulations of the present invention, uponcontact with body fluids, undergo gradual degradation. The life of abiodegrdable polymer in vivo depends upon, among other things, itsmolecular weight, crystallinity, biostability, and the degree ofcrosslinking. In general, the greater the molecular weight, the higherthe degree of crystallinity, and the greater the biostability, theslower biodegradation will be.

If a subject composition is formulated with a therapeutic agent or othermaterial, release of such an agent or other material for a sustained orextended period as compared to the release from an isotonic salinesolution generally results. Such release profile may result in prolongeddelivery (over, say 1 to about 2,000 hours, or alternatively about 2 toabout 800 hours) of effective amounts (e.g., about 0.0001 mg/kg/hour toabout 1 0 mg/kg/hour) of the agent or any other material associated withthe polymer.

A variety of factors may affect the desired rate of hydrolysis ofpolymers of the subject invention, the desired softness and flexibilityof the resulting solid matrix, rate and extent of bioactive materialrelease. Some of these factors include the selection/identity of thevarious subunits, the enantiomeric or diastereomeric purity of themonomeric subunits, homogeneity of subunits found in the polymer, andthe length of the polymer. For instance, the present inventioncontemplates heteropolymers with varying linkages, and/or the inclusionof other monomeric elements in the polymer, in order to control, forexample, the rate of biodegradation of the matrix.

To illustrate further, a wide range of degradation rates may be obtainedby adjusting the hydrophobicities of the backbones or side chains of thepolymers while still maintaining sufficient biodegradability for the useintended for any such polymer. Such a result may be achieved by varyingthe various functional groups of the polymer. For example, thecombination of a hydrophobic backbone and a hydrophilic linkage producesheterogeneous degradation because cleavage is encouraged whereas waterpenetration is resisted.

One protocol generally accepted in the field that may be used todetermine the release rate of any therapeutic agent or other materialloaded in the polymer matrices of the present invention involvesdegradation of any such matrix in a 0.1 M PBS solution (pH 7.4) at 37°C., an assay known in the art. For purposes of the present invention,the term “PBS protocol” is used herein to refer to such a protocol.

In certain instances, the release rates of different polymer systems ofthe present invention may be compared by subjecting them to such aprotocol. In certain instances, it may be necessary to process polymericsystems in the same fashion to allow direct and relatively accuratecomparisons of different systems to be made. For example, the presentinvention teaches several different means of formulating the polymeric,matrices of the present invention. Such comparisons may indicate thatanyone polymeric system releases incorporated material at a rate fromabout 2 or less to about 1000 or more times faster than anotherpolymeric system.

Alternatively, a comparison may reveal a rate difference of about 3, 5,7, 10, 25, 50, 100, 250, 500 or 750 times. Even higher rate differencesare contemplated by the present invention and release rate protocols.

In certain embodiments, when formulated in a certain manner, the releaserate for polymer systems of the present invention may present as mono-or bi-phasic.

Release of any material incorporated into the polymer matrix, which isoften provided as a microsphere, may be characterized in certaininstances by an initial increased release rate, which may release fromabout 5 to about 50% or more of any incorporated material, oralternatively about 10, 15, 20, 25, 30 or 40%, followed by a releaserate of lesser magnitude.

The release rate of any incorporated material may also be characterizedby the amount of such material released per day per mg of polymermatrix. For example, in certain embodiments, the release rate may varyfrom about 1 ng or less of any incorporated material per day per mg ofpolymeric system to about 500 or more ng/day/mg. Alternatively, therelease rate may be about 0.05, 0.5, 5, 10, 25, 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, or 500 ng/day/mg. In still otherembodiments, the release rate of any incorporated material may be 10,000ng/day/mg, or even higher. In certain instances, materials incorporatedand characterized by such release rate protocols may include therapeuticagents, fillers, and other substances.

In another aspect, the rate of release of any material from any polymermatrix of the present invention maybe presented as the half-life of suchmaterial in the matrix.

In addition to the embodiment involving protocols for in vitrodetermination of release rates, in vivo protocols, whereby in certaininstances release rates for polymeric systems may be determined in vivo,are also contemplated by the present invention. Other assays useful fordetermining the release of any material from the polymers of the presentsystem are known in the art.

D. Implants and Delivery Systems

In its simplest form, a biodegradable delivery system for a therapeuticagent consists of a dispersion or solution of a lapachone, optionallytogether with one or more other therapeutic agents, in a polymer matrix.In other embodiments, an article is used for implantation, injection, orotherwise placed totally or partially within the body, the articlecomprising the subject compositions. It is particularly important thatsuch an article result in minimal tissue irritation when implanted orinjected into vasculated tissue.

Biodegradable delivery systems, and articles thereof, may be prepared ina variety of ways known in the art. The polymer or composition may bemelt-processed using conventional extrusion or injection moldingtechniques, or these products may be prepared by dissolving in anappropriate solvent, followed by formation of the device, and subsequentremoval of the solvent by evaporation or extraction.

Once a system or implant article is in place, it should remain in atleast partial contact with a biological fluid, such as blood, internalorgan secretions, mucus membranes, cerebrospinal fluid, and the like toallow for sustained release of any encapsulated therapeutic agent.

6. Dosages and Formulations of the Subject Compositions

In most embodiments, the subject polymers will incorporate the lapachoneor other therapeutic agent in an amount sufficient to deliver to apatient a therapeutically effective amount as part of a prophylactic ortherapeutic treatment. The desired concentration of active compound inthe particle will depend on absorption, inactivation, and excretionrates of the drug as well as the delivery rate of the compound from thesubject compositions. It is to be noted that dosage values may also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions. Typically, dosing will be determinedusing techniques known to one skilled in the art.

Further, the amounts of bioactive substances will vary depending uponthe relative potency of the agents selected. Additionally, the optimalconcentration and/or quantities or amounts of any particular therapeuticagent may be adjusted to accommodate variations in the treatmentparameters. Such treatment parameters include the polymer composition ofa particular microsphere preparation, the identity of the therapeuticagent utilized, and the clinical use to which the preparation is put,e.g., the site treated, the type of patient, e.g., human or non-human,adult or child, and the nature of the disease or condition.

The concentration and/or amount of any therapeutic agent or otherencapsulated material for a given subject composition may readilyidentified by routine screening in animals, e.g., rats, by screening arange of concentration and/or amounts of the material in question usingappropriate assays. Known methods are also available to assay localtissue concentrations, diffusion rates from microspheres and local bloodflow before and after administration of therapeutic formulationsaccording to the invention. One such method is microdialysis, asreviewed by T. E. Robinson et al., 1991, MICRODIALYSIS IN THENEUROSCIENCES, Techniques, volume 7, Chapter 1. The methods reviewed byRobinson may be applied, in brief, as follows. A microdialysis loop isplaced in situ in a test animal. Dialysis fluid is pumped through theloop. When microspheres according to the invention are injected adjacentto the loop, released drugs are collected in the dialysate in proportionto their local tissue concentrations. The progress of diffusion of theactive agents maybe determined thereby with suitable calibrationprocedures using known concentrations of active agents.

In certain embodiments, the dosage of the subject invention may bedetermined by reference to the plasma concentrations of the therapeuticagent or other encapsulated materials. For example, the maximum plasmaconcentration (C_(max)) and the area under the plasma concentration-timecurve from time 0 to infinity may be used.

The polymers of the present invention may be administered by variousmeans, depending on their intended use, as is well known in the art. Forexample, if subject compositions are to be administered orally, it maybe formulated as tablets, capsules, granules, powders or syrups.Alternatively, formulations of the present invention may be administeredparenterally as injections (intravenous, intramuscular, orsubcutaneous), drop infusion preparations, or suppositories. Forapplication by the ophthalmic mucous membrane route, subjectcompositions may be formulated as eyedrops or eye ointments. Theseformulations may be prepared by conventional means, and, if desired, thesubject compositions may be mixed with any conventional additive, suchas a binder, a disintegrating agent, a lubricant, a corrigent, asolubilizing agent, a suspension aid, an emulsifying agent or a coatingagent.

In addition, in certain embodiments, subject compositions of the presentinvention maybe lyophilized or subjected to another appropriate dryingtechnique such as spray drying.

The subject compositions may be administered once, or may be dividedinto a number of smaller doses to be administered at varying intervalsof time, depending in part on the release rate of the compositions andthe desired dosage.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of a subject composition which may be combined with a carriermaterial to produce a single dose vary depending upon the subject beingtreated, and the particular mode of administration.

Methods of preparing these formulations or compositions include the stepof bringing into association subject compositions with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a subject composition with liquid carriers, or finelydivided solid carriers, or both, and then, if necessary, shaping theproduct.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of a subjectcomposition as an active ingredient. Subject compositions of the presentinvention may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the subject composition ismixed with one or more pharmaceutically acceptable carriers and/or anyof the following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using lactose or milk sugars, as wellas high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared using abinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the subject compositions, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, peanut,sunflower, soybean, olive, castor, and sesame oils), glycerol,tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters ofsorbitan, and mixtures thereof.

Suspensions, in addition to the subject compositions, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol, and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax, or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the appropriate body cavity and release theencapsulated therapeutic agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for transdermal administration include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches, andinhalants. A subject composition may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that may be required. For transdermaladministration, the complexes may include lipophilic and hydrophilicgroups to achieve the desired water solubility and transport properties.

The ointments, pastes, creams and gels may contain, in addition tosubject compositions, other carriers, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof. Powders and sprays may contain, in additionto a subject composition, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of such substances. Sprays may additionally contain customarypropellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane.

Microspheres which may be administered in inhalant or aerosolformulations according to the invention include agents, such asadjuvants, diagnostic agents, imaging agents, or therapeutic agentsuseful in inhalation therapy, which may be presented in a form which issoluble or substantially soluble in the selected propellant system.

The particle size of the particulate medicament should be such as topermit inhalation of substantially all of the medicament into the lungsupon administration of the aerosol formulation and will thus desirablybe less than 20 microns, preferably in the range 1 to 10 microns, e.g.,1 to 5 microns. The particle size of the medicament may be reduced byconventional means, for example by milling or micronisation.

The final aerosol formulation desirably contains 0.005-10% w/w,preferably 0.005-5% w/w, especially 0.01-1.0% w/w, of medicamentrelative to the total weight of the formulation.

It is desirable, but by no means required, that the formulations of theinvention contain no components which may provoke the degradation ofstratospheric ozone. In particular it is desirable that the formulationsare substantially free of chlorofluorocarbons such as CCl₃F, CCl₂F₂ andCF₃CCl₃. As used herein “substantially free” means less than 1% w/wbased upon the propellant system, in particular less than 0.5%, forexample 0.1% or less.

The propellant may optionally contain an adjuvant having a higherpolarity and/or a higher boiling point than the propellant. Polaradjuvants which may be used include (e.g., C₂₋₆) aliphatic alcohols andpolyols such as ethanol, isopropanol and propylene glycol, preferablyethanol. In general only small quantities of polar adjuvants (e.g.,0.05-3.0% w/w) may be required to improve the stability of thedispersion—the use of quantities in excess of 5% w/w may tend todissolve the medicament. Formulations in accordance with the inventionmay preferably contain less than 1% w/w, e.g. about 0.1% w/w, of polaradjuvant. However, the formulations of the invention are preferablysubstantially free of polar adjuvants, especially ethanol. Suitablevolatile adjuvants include saturated hydrocarbons such as propane,n-butane, isobutane, pentane and isopentane and alkyl ethers such asdimethyl ether. In general, up to 50% w/w of the propellant may comprisea volatile adjuvant, for example 1 to 30% w/w of a volatile saturatedC1-C6 hydrocarbon.

Optionally, the aerosol formulations according to the invention mayfurther comprise one or more surfactants. The surfactants must bephysiologically acceptable upon administration by inhalation. Withinthis category are included surfactants such as L-x-phosphatidylcholine(PC), 1,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitantrioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene(20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate,natural lecithin, oleyl polyoxyethylene (2) ether, stearylpolyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, blockcopolymers of oxyethylene and oxypropylene, synthetic lecithin,diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate,isopropyl myristate, glyceryl monooleate, glyceryl monostearate,glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethyleneglycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil,glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil.Preferred surfactants are lecithin, oleic acid, and sorbitan trioleate.

If desired, the surfactant may be incorporated into the aerosolformulation in the form of a surface coating on the particulatemedicament. In this case, the use of substantially non-ionic surfactantswhich have reasonable solubility in substantially non-polar solvents isfrequently advantageous since it facilitates coating of the medicamentparticles using solutions of surfactants in non-polar solvents in whichthe medicament has limited or minimal solubility.

The amount of surfactant employed in coating the particulate medicamentis desirably in the range 0.1 to 10% w/w preferably 1 to 10% w/w,relative to the medicament. Where the surfactant is present as a surfacecoating, the amount may advantageously be chosen such that asubstantially monomolecular coating of sent is formed. However, it ispreferable-that the formulations of the invention are substantially freeof surfactants, i.e., contain less than an effective stabilizing amountof a surfactant such as less than 0.0001% by weight of medicament.

The formulations of the invention may be prepared by dispersal of themedicament in the selected propellant and/or co-propellant in anappropriate container, e.g., with the aid of sonication. Preferably theparticulate medicament is suspended in co-propellant and filled into, asuitable container. The valve of the container is then sealed into placeand the propellant introduced by pressure filling through the valve inthe conventional manner. The active ingredient may be thus suspended ordissolved in a liquified propellant, sealed in a container with ametering valve and fitted into an actuator. Such metered dose inhalersare well known in the art. The metering valve may meter 10 to 500 μL andpreferably 25 to 150 μL. In certain embodiments, dispersal may beachieved using dry powder inhalers (e.g., spinhaler) for themicrospheres (which remain as dry powders). In other embodiments,nanospheres, may be suspended in an aqueous fluid and nebulized intofine droplets to be aerosolized into the lungs.

Sonic nebulizers may be used because they minimize exposing the agent toshear, which may result in degradation of the compound. Ordinarily, anaqueous aerosol is made by formulating an aqueous solution or suspensionof the polymeric materials together with conventional pharmaceuticallyacceptable carriers and stabilizers. The carriers and stabilizers varywith the requirements of the particular compound, but typically includenon-ionic surfactants (Tweens, Pluronics, or polyethylene glycol),innocuous proteins like serum albumin, sorbitan esters, oleic acid,lecithin, amino acids such as glycine, buffers, salts, sugars, or sugaralcohols. Aerosols generally are prepared from isotonic solutions.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Certain pharmaceutical compositions of this invention suitable forparenteral administration comprise one or more subject compositions incombination with one or more pharmaceutically acceptable sterileisotonic; aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be-reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity may be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Microsphere and/or nanosphere compositions may be suspended in apharmaceutically acceptable solution, such as saline, Ringer's solution,dextran solution, dextrose solution, sorbitol solution, a solutioncontaining polyvinyl alcohol (from about 1% to about 3%, preferablyabout 2%), or an osmotically balanced solution comprising a surfactant(such as Tween 80 or Tween 20) and a viscosity-enhancing agent (such asgelatin, alginate, sodium carboxymethylcellulose, etc.). In certainembodiments, the composition is administered subcutaneously. In otherembodiments, the composition is administered intravenously. Forintravenous delivery, the composition is preferably formulated asmicrospheres or nanospheres on average less than about 15 microns, moreparticularly less than about 10 microns, and still more particularlyless than about 5 microns in average diameter.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

1. Enhancement of Solubility and Bioavailability of β-Lapachone UsingCyclodextrin Inclusion Complexes

MATERIALS AND METHODS

Materials α-CD, β-CD, γ-CD and HPβ-CD were obtained from CyclodextrinTechnologies Development, Inc. (CTD) (High Springs, Fla.) with >98%purity. β-Lap was synthesized following a previously reported procedure(S. M. Planchon, S. Wuerzberger, B. Frydman, D. T. Witiak, P. Hutson, D.R. Church, G. Wilding and D. A. Boothman. Cancer Res. 55(17):3706-3711(1995)). Phosphate-buffered saline (PBS, pH=7.4) was purchased fromFisher Scientific (Pittsburg, Pa.). RPMI 1640 medium, fetal bovineserum, L-glutamine, penicillin and streptomycin were purchased fromHyclone (Logan, Utah) and Life Technologies, Inc. (Rockville, Md.).MCF-7 breast cancer cells were routinely passed at 1:5-1:20 dilutionsevery five days using mycoplasma-free 0.05% trypsin as described (J. J.Pink, S. M. Planshon, C. Tagliarino, S. M. Wuerzberger-Davis, M. E.Varnes, D. Siegel and D. A. Boothman. J Biol Chem. 275:5416-5424(2000)).

Phase Solubility Studies of CD.β-lap Inclusion Complexes

Solubility studies were performed by adding excess amount of β-lap to aseries of PBS buffer that contain different concentrations of each CDmolecule ranging from zero to its solubility limit (see Table 1 for thesolubility limit of each CD molecule). A schematic diagram of theintermolecular interactions and their effect on solubility is providedas FIG. 1. The suspensions were stirred at 25° C. until dissolutionequilibrium was reached. Then aliquots were withdrawn, filtered (Nylonsyringe filter 0.2 μm pore size from Fisher Scientific (Pittsburg, Pa.))and analyzed for β-lap concentrations by UV-Vis spectrophotometry(λ_(max)=257.2 nm, ε=109.6 ml/(mg·cm)). Phase solubility diagram foreach CD was obtained by plotting the β-lap solubility at dissolutionequilibrium as a function of the CD concentration. The associationconstant (K_(c)) $\begin{matrix}{K_{c} = \frac{Slope}{Y - {{intercept} \times \left( {1 - {Slope}} \right)}}} & (1)\end{matrix}$for the complex formation was calculated based on Equation 1 assuming a1:1 ratio of complex formation (T. Higuchi and K. A. Connors. Adv AnalChem Instrum. 4:117-212 (1965)).¹H NMR Study of CD.β-lap Inclusion Complexes

All ¹H NMR spectra were obtained on a Varian 600 MHz NMR spectrometer.The probe temperature was set at 25° C. ¹H-NMR spectrum of β-lap wasassigned by homonuclear correlation spectoscopy (COSY) and heteronuclearmultiple quantum coherence spectroscopy (HMBC). One dimensionalgradient-enhanced ROESY (GROESY) experiments were carried out by usingthe following pulse sequence; relaxation delay=1 s, 90° pulse width=8.2μs, spin lock time=400 ms and the acquisition time=3.495 s. Theconcentrations of β-lap and HPβ-CD for the GROESY experiments were 10.6and 58.8 mM in D₂O, respectively.

The complex for the NMR shift titration study was prepared by adding 78μl of β-lap stock solution (1.64 mM in MeOH). The solution was dried andthen variable amounts of β-CD and HPβ-CD solution in D₂O were added. Theresulting β-lap (0.123 mM) and β-CD (0.1-14.7 mM) or HPβ-CD (0.5-430 mM)solutions were vigorously stirred at 25° C. overnight to ensure thereaching of equilibrium. The association constants can be determinedbased on Equation 2 (A. Botsi, K. Yannakopoulou, B. Perly and E.Hadjoudis, J Org Chem. 60: 4017-4023 (1995)). $\begin{matrix}{{\Delta\delta}_{{Hc}\quad{or}\quad{Hd}} = \frac{K_{c}{{\Delta\delta}_{0}\left( {{{\Delta\delta}_{0}\lbrack{CD}\rbrack} - {{\Delta\delta}\left\lbrack {\beta\text{-}{Lap}} \right\rbrack}} \right)}}{{\Delta\delta}_{0} + {K_{c}\left( {{{\Delta\delta}_{0}\lbrack{CD}\rbrack} - {{\Delta\delta}\left\lbrack {\beta\text{-}{Lap}} \right\rbrack}} \right)}}} & (2)\end{matrix}$For methyl protons (Hc) on β-lap, Δδ_(HC) denotes the difference ofchemical shift between the two splitting methyl groups at a particularconcentration of CD. For aromatic Hd protons, Δδ_(Hd)(Δδ_(Hd)=7.787−δ_(i)) is calculated as the difference between thechemical shift of pure β-lap (7.787 ppm) from that of CD.β-lap inclusioncomplex at a particular concentration of CD (δ_(i)). For both Hc and Hdprotons, Δδ₀ denotes the difference between pure β-lap and pure CD.β-lapinclusion complexes, [CD] stands for the concentration of cyclodextrinand [β-lap] denotes the concentration of β-lap used in this experiment(0.123 mM).Fluorescence Study of CD.β-lap Inclusion Complexes

A fluorescence study was preformed on a LS45 Luminescence Spectrometer(Perkin Elmer Instruments) with 100 nm/min scan speed and 10 nm for bothexcitation and emission slit widths. Initially, emission spectra ofβ-lap (0.015 mg/ml) in PBS buffer were obtained at different excitationwavelengths to determine the optimal values of λ_(ex) and λ_(em) forspectrophotometry measurements. The effect of CD concentrations on thefluorescence spectra of β-lap was studied. In these studies, each samplewas prepared by adding the same volume (4 ml) of a stock solution ofβ-lap (0.005 mg/ml) but different quantities of CD inside a 5 mlvolumetric flask filled with PBS buffer. The resulting solutions werevigorously stirred at 25° C. overnight to ensure the reaching ofequilibrium. Emission spectra of β-lap at different CD concentrationswere obtained at λ_(ex)=330 nm. The fluorescence intensity at λ_(em)=436nm was measured and used to determine the value of K_(c) of CD.β-lapinclusion complex.

In vitro Cytotoxicity Assays

The cytotoxicity of β-CD.β-lap and HPβ-CD.β-lap inclusion complexes toMCF-7 breast cancer cells was determined following a previouslypublished procedure (16). The MCF-7 cells were grown in RPMI 1640 mediumsupplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 units/mlpenicillin, and 100 mg/ml streptomycin. In cytotoxicity studies, cellswere first seeded into ninety-six well plates at 1×10⁴ cells/well in 1ml medium and allowed to attach overnight. Media were removed 24 hlater, and new medium (1 ml) containing different concentrations of CDalone or β-lap in CD inclusion complex were added to each well. After 4h, the media were removed and replaced with drug-free growth media.Cells were allowed to grow for an additional 6 days. On day 7, cellswere washed with PBS after media removal, and 250 μl double distilledMilli Q H₂O was added to each well. After one freeze-thaw cycle, TNEbuffer (2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) with 10 μg/mlHoechst 33258 (Sigma) fluorescent dye was added to each well. Changes incell number, measured as DNA content, were then determined by anadaptation of the method of Labarca and Paigen (C. Labarca and K.Paigen. Anal Biochem. 102:344-352 (1980)) and analyzed with a PerkinElmer HTS 7000 Bio Assay Reader with excitation wavelength of 360 nm andemission wavelength of 460 nm. Data were expressed as relative growth(T/C) by dividing DNA content of treated cells (T) by that of untreatedcells (C) at identical times. The reproducibility of each data point isrepresented by the means, ±SEM, from at least six replicate wells. β-Lapin dimethylsulfoxide (DMSO) was used as a positive control to comparethe drug cytotoxicity to MCF-7 cells.

Animal Toxicity Studies

C57Blk/6 female mice (3-4 week-old, 18-20 g) (Jackson Labs, Me.) wereused to study the morbidity and mortality of mice treated withHPβ-CD.β-lap inclusion complex. Four mice per group were used for eachdose, which varied from 20 to 100 mg/kg. Two groups of four mice wereused for 60 mg/kg, since this dose proved to be near the LD₅₀ (lethaldose that kills 50% of the mice population) of the β-lap in HPβ-CDinclusion complex. Mice were injected (i.p.) every Monday, Wednesday andFriday for three weeks for a total of 10 injections. Control animals (4mice/group) were injected with 5000 mg/kg of HPβ-CD alone to evaluateits toxicity. This HPβ-CD dose is approximately ten times of the HPβ-CDamount introduced at the highest dose of β-lap (100 mg/kg) via theHPβ-CD.β-lap inclusion complex. The higher dose of HPβ-CD was used toensure the lack of toxicity of this compound. Weight and lethality weremeasured on a daily basis following initial drug administration. Allanimals were maintained in a facility accredited by the Association forAssessment and Accreditation of Laboratory Animal Care according to the“Principles of Laboratory Animal Care” of the National Institutes ofHealth.

RESULTS AND DISCUSSION

Solubility Study

The effect of cyclodextrins on the aqueous solubility of β-lap wasevaluated using the phase solubility method (T. Higuchi and K. A.Connors. Adv Anal Chem Instrum. 4:117-212 (1965)). FIG. 2 shows thephase diagrams of β-lap with four different types of CDs in PBS buffer.The solubility of β-lap increased linearly as a function of α-, β-, orHPβ-CD concentrations. These phase diagrams are classified as type A_(L)by Higuchi, which denotes a linear increase in solubility. In contrast,γ-CD showed a typical B_(S)-type solubility curve, which denotes aninitial rise in the solubility of the solute followed by a plateau anddecreased region due to the limited solubility of the complexes.

Increases in β-lap solubility in aqueous CD solutions are consistentwith the formation of inclusion complexes between β-lap and CDmolecules. In general, the main driving force for the complex formationis the hydrophobic interactions between a poorly soluble guest compound,such as β-lap, and the apolar cavity of the. CD molecule. Thehydrophobicity. and geometry of the guest molecule as well as the cavitysize of the CD molecule are important parameters for the complexformation. In the current study, the enhancement of β-lap solubility ishighly dependent on the type of CD molecule. For example, the phasediagram for β-CD shows a much higher slope (0.16) than that of α-CD(0.0035) and the linear region ([γ-CD]<20 mM) of γ-CD (0.024,FIG. 2A),demonstrating that β-CD is more effective to solubilize β-lap. Based onthe phase solubility diagrams, the association constants for thedifferent inclusion complexes are determined using Equation 1. Thevalues of K_(c) are 20.0±0.7, (1.23±0.01)×10³, (0.94±0.08)×10³ and 160±5M⁻¹ for α-CD, β-CD, HPβ-CD, and γ-CD, respectively.

The different association constants for different cyclodextrin moleculesindicate the importance of cavity size to encapsulate the β-lapmolecule. α-CD has the lowest affinity to associate with β-lap,presumably because β-lap cannot fit into the relatively smallhydrophobic cavity of α-CD (diameter ˜5 Å, Table 1). This is inagreement with other studies (M. V. Rekharsky and Y. Inoue. Chem Rev.98:1875-1917 (1998)) in which guest molecules carried a phenyl moiety.On the other hand, although the wider cavity size of γ-CD (diameter ˜8Å) allows room for encapsulation (K_(c) increased by a factor of 8 forγ-CD over α-CD), it has lower affinity to associate with β-lap than thatof β-CD and HPβ-CD, which have smaller cavity size. Therefore, β-CD andHPβ-CD appear to be significantly better host molecules for β-lapencapsulation. The much higher association constants of HPβ-CD and β-CDshow the importance of appropriate cavity size in facilitating theinteractions between β-lap and HPβ-CD or β-CD, a s further supported bymolecular recognition studies of host-guest chemistry (K. A. Connors.Chem Rev. 97:1325-1357 (1997)).

Even though β-CD is a better host molecule for β-lap than α-CD and γ-CD,its application to maximize the solubility of β-lap is limited by thesolubility of β-CD vehicle itself (16.3 mM). Consequently, the maximalsolubility of β-lap in β-CD solution is limited to 2.8 mM or 0.68 mg/ml.This concentration is still relatively low for systemic administrationsof this drug. To overcome this problem, we used HPβ-CD molecule as a‘3-lap carrier. HPβ-CD is formed by covalent modification of theexternal hydrox group on β-CD by hydroxylpropyl groups. The modificationsignificantly increased the solubility limit of HPβ-CD (360 mM, a factorof 22 over β-CD). The maximal solubility of β-lap in HPβ-CD solutionreached 66.0 mM or 16.0 mg/ml, a 24-fold increase over that in β-CDvehicle and a 413-fold increase over β-lap aqueous solubility (0.16 mM).HPβ-CD provides the most effective solubilization of β-lap.

NMR Study of CD.β-lap Inclusion Complexes

NMR spectroscopy is a powerful tool to study the inclusion phenomena. Ithas been shown that GROESY spectroscopy can be used to accurately detectthe nuclear overhauser effect (NOE) (P. Adell, T. Parella, F.Sanchez-Ferrando and A. Virgili. J Magn Reson. 108: 77-80 (1995);Y.Ikeda, S. Motoune, T. Matsuoka, H. Arima, F. Hirayama and K. Uekama. JPharm Sci. 91(11): 2390-2398 (2002)). In this study, we carried out theGROESY experiment to gain insight regarding the molecular structure ofHPβ-CD.β-lap inclusion complex. FIG. 3 shows the GROESY spectra of theHPβ-CD.β-lap inclusion complex obtained by exciting every proton ofβ-lap (Ha to Hg). The significant NOE enhancement of the H5 and H3protons located inside the HPβ-CD cavity was observed with the selectiveexcitation of the Hc protons from β-lap. In contrast, no obvious NOEenhancement was observed with the selective excitation of the rest ofβ-lap protons, suggesting that the methyl moiety of β-lap is boundinside the cavity. This result also suggests that HPβ-CD forms a. 1:1inclusion complex with β-lap.

It is well known that the insertion of a guest molecule into thehydrophobic cavity of cyclodextrin can affect chemical shifts of theguest protons. In this experiment, we studied the effect of β-CD andHPβ-CD on the resonance of β-lap protons. FIG. 4A shows the effect ofincreasing β-CD concentration on the ¹H NMR spectra of phenyl protons ofβ-lap. Interestingly, Hd was the only proton that showed upfield shiftsas a result of increasing β-CD concentrations. Above [β-CD]=11.4 mM, nofurther changes of the upfield shift were observed (data not shown). Theupfield shifts of Hd as a result of increasing of HPβ-CD concentrationswere also found. FIG. 4B shows the effect of β-CD on the ¹H NMR spectraof methyl (Hc) and methylene (Ha, Hb) protons of β-lap. A splitting ofthese three groups of proton peaks was observed due to the formation ofinclusion complex. This effect was most pronounced with the methylprotons (Hc) whereas Ha protons had the least effect, suggesting theformation of diastereomeric complexes between β-lap and CD. A splittingof Ha, Hb and Hc was also found with HPβ-CD, but the signal wasinterfered with by the methylene protons from hydroxypropyl groups onHPβ-CD.

The upfield shift of Hd (not for other phenyl protons) and thesplittings of Ha, Hb and Hc indicate that these changes are the resultof inclusion complex formation but not due to the non-specificinteraction between cyclodextrin and β-lap. Chemical shift changes of Hd(FIG. 5A) as a function of β-CD and BPβ-CD concentrations and thesplitting of Hc (FIG. 5B) as a function of β-CD gave good fits with a1:1 complex model as shown in Equation 2 (A. Botsi, K. Yannakopoulou, B.Perly and E. Hadjoudis, J Org Chem. 60: 4017-4023 (1995)). Theassociation constants determined from these data are 774±52 M⁻¹ (Hdshift), 734±20 M⁻¹ (Hc splitting) for β-CD.β-lap inclusion complex, and662±27 M⁻¹ (Hd shift) for Hβ-CD.β-lap inclusion complex.

Fluorescence Studies of β-lap Inclusion Complex

In the course of this study, we discovered that β-lap was a fluorescentmolecule, and we used fluorescence spectroscopy to further study theassociation of HPβ-CD.β-lap and β-CD.β-lap inclusion complexes. FIG. 6Ashows a series of emission spectra of β-lap alone in PBS buffer atdifferent excitation wavelengths ranging from 257 to 360 nm. These datashowed that an excitation wavelength at 330 nm gave the highest emissionintensity. For all the excitation wavelengths, the maximum emissionwavelength was located at 436 nm. These experiments established theoptimal spectroscopy conditions for β-lap complexation studies(λ_(ex)=330 nm, λ_(em)=436 nm).

FIG. 6B shows the dependence of β-lap emission spectra as a function ofHPβ-CD concentrations in PBS buffer. All the experiments were carriedout at the same excitation wavelength (λ_(ex)=330 nm) and same β-lapconcentration (18 μM). Results showed that the β-lap emission intensitydecreased when the HPβ-CD concentration increased (FIG. 6B). Inaddition, there is a slight blue shift (˜6 nm) of the maximum emissionwavelength in solution containing HPβ-CD. The change in fluorescenceintensity and maximum emission wavelength of guest β-lap compound byaddition of cyclodextrins is another indication of the formation ofinclusion complexes between these two compounds. Upon encapsulationinside the hydrophobic cavity of CD molecules, the β-lap compoundencounters a different chemical environment compared to aqueoussolution. Geometric restrictions due to space limitations in the CDcavity and reduced polarity due to the hydrophobic cavity of CD arefound to alter the energetics and dynamics of the photophysical andphotochemical processes of the guest molecule (V. Ramamurthy, D. F.Eaton. Acc Chem Res. 21:300-306 (1988)). The blue shift is consistentwith the fact that β-lap experiences a less polar environment in thehydrophobic cavity of HPβ-CD.

Next, we determined the association constant for the formation ofinclusion complex based on the fluorescence data. Emission intensity at436 nm was used for these studies. Scatchard analysis by Equation 3 (E.E. Sideris, G. N. Valsami, M. A. Koupparis and P. E. Macheras, PharmRes. 9(2): 1568-1574 (1992)) was used to determine the associationconstant (K_(c)) of the inclusion complex.R/[CD] _(f) =n K _(c) −RK _(c)   (3)where [CD]_(f) is the unbound (free) molar concentration of CD, n is thenumber of binding sites i.e. the stoichiometry of the complex and R isthe molar fraction of β-lap bound to CD.

The values of K_(c) are (1.10±0.06)×10³ M⁻¹ (R²=0.97) and(1.06±0.06)×10³ M⁻¹ (R²=0.98) for β-CD.β-lap and HPβ-CD.β-lap complexes,respectively. The numbers of binding sites (n) of β-CD.β-lap andHPβ-CD.β-lap inclusion complexes were found to be 1.04±0.02 and1.01±0.02, respectively, which confirm the formation of 1:1 inclusioncomplexes. The values of K_(c) from fluorescence measurement areconsistent with those from phase solubility studies, but are higher thanthe data from NMR measurement. This different is most likely due todifferent solvents (e.g., PBS buffer were used in fluorescence and phasesolubility studies, in comparison to D₂O in NMR studies).

In vitro Cytotoxicity Studies in MCF-7 Cells

In order to evaluate the biological activity of β-lap when it forms aninclusion complex with cyclodextrin, initial cytotoxicity DNA assaysusing MCF-7 human breast cancer cells were performed. Previous studies(J. J. Pink, S. M. Planshon, C. Tagliarino, S. M. Wuerzberger-Davis, M.E. Varnes, D. Siegel and D. A. Boothman. J Biol Chem. 275:5416-5424(2000); S. M. Wuerzberger, J. J. Pink, S. M. Planchon, K. L. Byers, W.G. Bornmann and D. A. Boothman. Cancer Res. 58(9):1876-85 (1998); C.Tagliarino, J. J. Pink, G. R. Dubyak, A. L. Nieminen and D. A. Boothman.J Biol Chem. 276(22):19150-9 (2001)) have demonstrated thatNQO1-expressing MCF-7 cells treated under these conditions tested notonly growth inhibition, but the results can be equated to loss ofsurvival using colony forming ability assays. Log-phase MCF-7 cells wereexposed to different concentrations of β-lap in HPβ-CD inclusioncomplex, β-lap in β-CD inclusion complex, or with HPβ-CD and β-CD alonefor four hours. Drugs were then removed and DNA content as a measure ofcell survival was determined. β-Lap in DMSO was used as a positivecontrol for comparison. FIG. 6 shows the viability of MCF-7 cellsexposed to HPβ-CD.β-lap, β-CD.β-lap inclusion complexes, or with HPβ-CDand β-CD alone. The primary x-axis is the β-lap concentration used inthis experiment and the secondary x-axis is the concentration of HPβ-CDand β-CD required to solubilize β-lap. Cell viability of MCF-7 cells wasstatistically identical for cells treated with vehicles (HPβ-CD, β-CD)alone or with PBS for 4 hours. These data showed that pure HPβ-CD (0 to18.8 μM) and β-CD (0 to 20.8 μM) alone showed no cytotoxicity or growthinhibition. β-lap in HPβ-CD and β-lap in β-CD inclusion complexes showedsimilar cytotoxic responses for the entire range of β-lap-equivalentdoses (FIG. 7). Quantitatively, the drug potency was measured as TD₅₀,the toxic dose that kills 50% of the cell population. The TD₅₀ values ofβ-lap in HPβ-CD and β-CD inclusion complexes were found to be the sameat 2.1 μM for a 4 h transient drug exposure. These values were slightlyhigher than that from β-lap in DMSO, whose TD₅₀ value is 1.7 μM.

In vivo Analyses of β-lap Toxicity

To evaluate the bioavailability of β-lap in CD inclusion complexes,C57Blk/6 mice were injected with increasing concentrations of β-lap inHPβ-CD inclusion complex three days per week for three weeks, andchanges in weight and survival were recorded. Results showed nomorbidity (decreases in weight loss) or lethality of mice for controlgroup injected i.p. with vehicles alone, or for mice injected i.p with20 to 50 mg/kg of β-lap in HPβ-CD inclusion complex. In contrast, miceinjected i.p. with 70 to 100 mg/kg showed both morbidity and 100%lethality (FIG. 8). Finally, mice treated with 60 mg/kg β-lap in HPβ-CDinclusion complex i.p. resulted in significant morbidity (loss of >15%body weight in most animals) and lethality (⅞ animals died within 45days of the treatment regimen). Consequently, the LD₅₀ (lethal dose thatkills 50% of mice population) value of β-lap in HPβ-CD inclusion complexwas estimated to be between 50-60 mg/kg in 18-20 gram C57Blk/6 mice.This was determined by considering that 50 mg/kg kills 0% of micepopulation and 60 mg/kg kills 85% of mice population in the course ofthis experiment. Interestingly, mice responded to doses above 50 mg/kgβ-lap in HPβ-CD inclusion complex, but not with HBβ-CD vehicle alone,with unusual but temporary drug reactions. Within 15 minspost-i.p.-injection, mice were observed to have a shivering reflex anddifficulty in breathing. These drug responses lasted approximately twohours, with mice exposed to 40-50 mg/kg recovering completely withessentially no weight loss noted overtime. In contrast, most miceexposed to >60 mg/kg exhibited similar drug responses that resulted inlethality. Preliminary autopsies with mice that ultimately died did notresult in the detection of major damage to vital organs, and moredetailed analyses of cause of death are ongoing. Our studies indicate anearly 3-fold greater bioavailability of β-lap in vivo compared toprevious animal studies using Cremophor as a vehicle for β-lapadministration, where an LD₅₀ of >150 mg/kg was reported (C. J. Li, Y.Z. Li, A. V. Pinto and A. B. Pardee. Proc Natl Acad Sci. USA.96(23):13369-74 (1999)).

CONCLUSION

Phase solubility studies of β-lap in complexation with α-CD, β-CD,HPβ-CD or γ-CD were carried out to overcome the problems of β-lapsolubility and bioavailability. Hβ-CD demonstrated the maximumenhancement of β-lap solubility to 16.0 mg/ml or 66.0 mM, more than a400-fold increase over β-lap solubility in water (0.04 mg/ml or 0.16mM). The association constants of β-lap with cyclodextrins weredetermined by the phase solubility method, ¹H NMR and fluorescencespectroscopy (λ_(ex)=330 nm, λ_(em)=436 nm). β-CD and HPβ-CD showedhigher binding affinity (K_(c)=0.9−1.2×10³ M⁻¹) to β-lap than α-CD (20M⁻¹) and γ-CD (160 M⁻¹ ). Cytotoxicity assays indicated littledifferences in biological activity between β-lap in HPβ-CD or β-CDinclusion complexes, with nearly identical cell responses (cell death ininduced apoptosis) and TD₅₀ values (2.1 μM). Finally, studies ofmorbidity and mortality in C57Blk/6 mice suggested a LD₅₀ between 50-60mg/kg, with no morbidity or mortality following 20-50 mg/kg β-lap inHPβ-CD inclusion complex. Complexation of β-lap with HPβ-CD offers amajor advancement in improvement of bioavailability of this very activeanticancer agent.

MDA_MB-468 NQO1+Tumor (468) cells were injected into the right and leftflanks of 18-20 gm athymic nude mice and tumors of 20-50 mm³ volume wereallowed to grow in 20-30 days. When tumor volumes reached 20-30 mm³ (atday 26), mice were then treated (by intraperitoneal injection) everyother day with the indicated doses (in mg/kg) of β-lap (55-70 mg/kg weretested). The maximum tolerated dose of β-lap in athymic nude mice wasdetermined to be 80 mg/kg). Ten (10) β-lap doses were given every otherday, and data are shown for only one cycle of β-lap treatment. Multiplecycles of treatments are now being tested, as are various dose regimenand scheduling protocols. For the 468 cell line, significant antitumorresponses were observed with β-lap in a dose-responsive fashion (FIG.9). Significant antitumor responses were observed with 65 and 70 mg/kg.

2. Lung Cancer Therapy

β-lap activity against human and mouse NSCLC. As expected forNQO1-expressing cell lines, β-lap treatment caused lethality andapoptosis in CC-10 or A549 NSCLC cells, which was blocked by 50 μMdicoumarol (FIG. 10). As observed with β-lap-treated NQO1-expressinghuman breast or prostate cancer cells (Pink, J. J., Planchon, S. M.,Tagliarino, C., Vames, M. E., Siegel, D., and Boothman, J Biol Chem,275: 5416-5424, 2000; Planchon, S. M., Pink, J. J., Tagliarino, C.,Bornmann, W. G., Varnes, M. E., and Boothman, D. A. Experimental CellResearch, 267: 95-106, 2001), atypical PARP cleavage was observed afterβ-lap exposure, and this apoptotic proteolysis was diminished bydicoumarol (FIG. 10B). PARP cleavage to a 60 kDa fragment and specificp53 proteolysis are diagnostic for β-lap-induced μ-calpain-mediatedapoptosis. These data are consistent with our hypothesis that β-lapcauses lethality in an NQO1-dependent manner to cause μ-calpainapoptotic cell death. Similar results were observed in mouse cells:Lewis lung carcinoma (LLC), CC-10 tumor cell lines derived from aspontaneous mouse CC-10 lung cancer model, and L1C2 cells expressedelevated levels of NQO1 compared to normal lung tissue (Table 2), andwere extremely sensitive to β-lap (4 μM, 4 h). As expected,co-administration of dicoumarol prevented β-lap cytotoxicity in thesecells.

Evaluation of NQO1 levels in normal mouse CC-10 lung v. CC-10spontaneous tumors. In order to evaluate the efficacy of variousvehicles carrying β-lap under various routes of administration, it isessential that the proposed CC-10 or LLC model systems mimic humandisease with respect to NQO1 over-expression. To monitor NQO1 levels invivo, CC-10 mice with spontaneous tumors were sacrificed and NSCLC aswell as normal lung tissue was extracted and analyzed for NQO1activities as previously described (Table 2). Human A549 and variousmouse NSCLC cell lines were also evaluated (Table 2). As shown in Table2, NQO1 levels were elevated in mouse CC-10 tissue 8-fold compared toadjacent normal tissue. Furthermore, mouse NSCLC cell lines were alsoelevated 12- to 21-fold above normal tissue. Interestingly, the A549NSCLC cell line was also elevated over 1200-fold above normal mouse lungtissue (Table 2). TABLE 2 Elevation of NQO1 in CC-10 tumor and NSCLCcell lines vs. normal tissue. NQO1 enzymatic activities were measuredfrom spontaneous CC-10 lung tumor tissue v. adjacent normal lung tissueas described (Pink, J. J., Planchon, S. M., Tagliarino, C., Varnes, M.E., Siegel, D., and Boothman, D. A. J Biol Chem, 275: 5416-5424, 2000).In adddition, mouse NSCLC cell lines, as well as the human A549 NSCLCcell line, were evaluated and compared to normal mouse tissue. Comparedto normal mouse lung tissue, CC-10 tumor tissue NQO1 activity waselevated 8-fold. NQO1 Activity X-Fold Above Cell Line/Tissue (μM CytoCreduced/

protein) Normal Normal Lung 4 1 (Mouse) CC-10 Tumor 34 8 (Mouse) CC-10Cell Line 88 21 (Mouse) L1C2 Cell Line 54 12 (Mouse) LLC Cell Line 75 18(Mouse) A549 Cell Line 5056 1200 (Human)

Development of β-lap-loaded microspheres. Development of microspheresable to release β-lap under controlled conditions, and specificallywithin or near the lung, is a major goal of this grant. In initialexperiments, the loading density of β-lap was controlled at 2% inpolymer microspheres. Polymer microspheres were synthesized using asingle-emulsion procedure (see Methods in Deng, X. M., Xiong, C. D.,Cheng, L. M., Huang, H. H., and Xu, R. P. Journal of applied polymerscience, 55: 1193-1196, 1995). Scanning electron microscopy (SEM)imaging of β-lap-loaded microspheres showed an average diameter of 3 μmof drug-carrying microspheres, a size that can be easily varied, andthat should accumulate in the lungs of treated mice.

Release studies. β-Lap release studies from polymer microspheres (n=3)were performed in PBS at 37° C. (FIG. 11). The time for 50% drug releaseof β-lap was 9 h, while nearly all (98%) was released in one week.Microsphere size can be varied (together with β-lap loading density) tocontrol β-lap release kinetics from microspheres. The needed therapeuticdose of β-lap is calculated to be about 0.035 mg. This dose permitsreaching the high-end of a therapeutic concentration of β-lap (10 μM) ina tumor with 3 cm diameter. Based on results in FIG. 11, microspheresystems can release therapeutic levels of β-lap in less than 5 h. Theseresults indicate that desirable brief, but elevated, delivery oftherapeutic doses of β-lap to primary and possibly metastatic tumorcells can be achieved with such microspheres.

Quantitative analyses of β-lap by HPLC-ESI-MS. To evaluate the in vivopharmacokinetics of β-lap delivery from polymer microspheres, it wasnecessary to develop an accurate, highly sensitive, and quantitativemethod to measure the plasma concentration-time relationships and tissuedistribution of β-lap. High pressure liquid chromatography-electrosprayionization-mass spectrometry (HPLC-ESI-MS) methodology for thequantitative analyses of β-lap was developed. The chromatographic mediumwas Ansys Metachem Polaris C18A (3 μm particle diameter) containedinside a column of 0.46 cm i.d.×5 cm in length. The isocratic mobilephase was 25 mM ammonium formate+acetonitrile (v/v=50/50), at a flowrate of 0.5 mL/min. β-Lap was detected by selective reaction monitoring,of the transition from 243 m/z (M+H)⁺ to 187 m/z. Preliminary datashowed superb sensitivity of detection under current experimentalconditions. The lowest limit of quantification was 1.1×10⁻¹⁴ mol ofinjected β-lap with a signal to noise ratio of 4:1. For a biologicalsample volume of 100 μL, this sensitivity permits detection of β-lap at0.1 nM (10⁻¹⁰ M). This methodology is suitable for quantitative analysesof β-lap levels in vivo. These data show the feasibility of HPLC-ESI-MSfor highly sensitive and quantitative analyses for β-lap.

3. Treatment of Prostate Cancer

Fabrication of β-lap-loaded polymer millirods. The extremely lowsolubility of β-lap in water has greatly limited the clinical use ofthis compound. The development of drug-impregnated millirods, whereinβ-lap release kinetics may be accurately permits the delivery of anefficacious form of therapy against human prostate tumors that commonlyover-express the NQO1 oxidoreductase. The extinction coefficient forβ-lap in water was determined to be 2.6×10⁴ cm⁻¹M⁻¹ at 257 nm wavelengthby UV-Vis spectrophotometry. Solubility experiments were performed byimmersing β-lap in PBS over a period of six days in an orbital shaker at37° C. At the start of the experiment the drug suspension was sonicatedfor 20 seconds to facilitate the fast reach of solubility equilibrium.Samples were taken and filtered by 0.2 μm Millipore filters via syringeto remove non-dissolved drug particles and diluted to accommodate theUV-Vis sensitive range before collecting absorbance data. Using theextinction coefficient, the solubility of β-lap in PBS buffer was2.6×10⁻⁴ M (0.05 mg/cc). This concentration is too low for i.v.administration in clinical applications. Preliminary experiments havedemonstrated the feasibility of controlling the loading density of β-lapat 10% in PLGA polymer millirods. Polymer millirods were fabricatedusing a compression heat molding procedure as previously developed inour lab. (Halpert, B., Sheehan, E. and Schmalhorst, W. (1998) Cancer 82,737-742.) Briefly, β-lap particles were mixed with polymer microspheres(4 μm), and various concentrations of glucose. Polymer microspheres werefabricated using a single emulsion procedure. Glucose particles wereintroduced as an excipient molecule at different loading densities (20%,40%) to control the release kinetics of β-lap. The solid particlemixture was placed in a Teflon tube and heated to 90° C. at acompression pressure of 40 atm for 2 h. After compression heat molding,the polymer millirods were removed and cooled to room temperature forfurther characterization.

Release studies. Release studies of β-lap were performed in PBS at 37°C. FIG. 12 shows the release profiles of β-lap from polymer millirods.Addition of glucose effectively controlled the release rate of β-lap.Increasing the percentage of glucose led to faster release kinetics ofβ-lap from millirod polymers. For example, the amount of released β-lapincreased from 0.05 mg in a millirod with 0% glucose to 0.5 mg in amillirod with 40% glucose after 7 h. The increased release rate was dueto the dissolution of glucose particles from the polymer matrix thatresults in pores and channels facilitating release of β-lap. However,β-lap may not have been intact after polymer construction and release.FIG. 13 shows the stacked UV-Vis spectra of β-lap released at differenttime points. Each spectrum was normalized to the peak UV absorbance andcompared to that of the original sample. Identical UV-Vis spectrastrongly suggest that structural integrity of the β-lap is maintainedthroughout the fabrication and controlled release studies. The necessarytherapeutic dose of β-lap is 0.035 mg, a local concentration that wouldachieve 10 μM in a 3 cm diameter tumor. Based on results in FIG. 12, allmillirods produced will release therapeutic levels of β-lap in less than5 h. TABLE 3 Dicoumarol blocks β-lap-induced apoptosis in NQO1⁺, but notNQO1⁻, isogenic LNCaP cells. Apoptosis (%) Cell Line After DrugTreatment Campto- (+Pretreated NQO1 β-Lapachone thecin Blocking Agent)Activity¹ DMSO (10 μM) (10 μM) LNCaP 3.0 ± 0.4  1.2 ± 0.7 28 ±7 30 ± 5LNCaP + DC² ND³ 0.91 ± 0.3 28 ±3 33 ± 9 LNCaP + DVED 3.9 ± 0.3 0.62 ±0.1 24 ±6 10 ± 3 DU-145 500 ± 48  0.08 ± 0.01 71 ±13 30 ± 6 DU-145 + DCN-D 0.10 ± 0.04 0.12 ±0.03 43 ± 12 DU-145 + DVED 502 ± 41  0.11 ± 0.0576 ±12 10 ± 5 PC-3 740 ± 100 0.06 ± 0.006 82 ±15 23 ± 3 PC-3 + DC N-D0.04 ± 0.002 0.04 ±0.005 22 ± 7 PC-3 + DVED 810 ± 130 0.03 ± 0.001 91±12  6 ± 5¹NQO1 activity, nmols cytochrome C reduced/min/mg protein;+DC², 50 μM dicoumarol;N-D³, no enzyme activity detected; TUNEL assays were used to monitorapoptosis, ±50 μM dicoumarol. Drug treatments were for 4 h, and TUNELassays performed 48 h post-treatment (Pink, J. J., Planchon, S. M.,Tagliarino, C., Varnes, M. E., Siegel, D. and Boothman, D. A. (2000) JBiol Chem 275, 5416-5424). Experiments were performed at least threetimes in duplicate.

Evidence that NQO1 ‘bioactivates’ β-lap in human CaP cells. Structuralsimilarities between β-lap and other naphthoquinones suggested that NQO1may be involved in its activation or detoxification. The IR-induction ofNQO1 was consistent with this compound's ability to sensitize IR-treatedcells. DU-145 or PC-3 cells were sensitive to β-lap in the absence ofIR. LNCaP cells, which lack NQO1 expression and activity (Table 3) wereresistant. Dicoumarol enhanced the survival of β-lap-treated DU-145 orPC-3 cells, increasing LD₉₀ values>3-fold for DU-145 and PC-3 cells,respectively; e.g., >95% lethality was noted in DU-145 cells after 4 μMβ-lap, whereas the drug was ineffective (>95% survival) with 50 μMdicoumarol. Dicoumarol had no influence on the survival ofβ-lap-resistant LNCaP cells, and β-lap-treated LNCaP cells exhibited3-fold less apoptosis than DU-145 or PC-3 cells (Table 3) (Planchon, S.M., Wuerzberger, S., Frydman, B., Witiak, D. T., Hutson, P., Church, D.R., Wilding, G. and Boothman, D. A. (1995) Cancer Res 55, 3706-3711).Changes in apoptosis (Table 3) mimicked lethality after drug exposure(X). In contrast, dicoumarol did not affect the lethality or apoptosisof LNCaP, DU-145 or PC-3 cells after camptothecin (CPT, a DNATopoisomerase I poison) exposures (Wuerzberger, S. M., Pink, J. J.,Planchon, S. M., Byers, K. L., Bornmann, W. G. and Boothman, D. A.(1998) Cancer Res 58, 1876-1885). DVED, an inhibitor of caspase-mediatedapoptosis, blocked CPT-mediated apoptosis (Table 3), but not loss ofsurvival (Pink, J. J., Wuerzberger-Davis, S., Tagliarino, C., Planchon,S. M., Yang, X., Froelich, C. J. and Boothman, D. A. (2000) Exp Cell Res255, 144-155.

β-Lap-mediated proteolysis during apoptosis. Cleavage ofpoly(ADP-ribosyl)polymerase (PARP) is a marker of apoptosis.Caspase-mediated cleavage results in an 89 kDa polypeptide by Westernblot analyses. Human CaP cells treated with β-lap exhibited an atypical˜60 kDa PARP cleavage fragment. As expected, CPT induced acaspase-mediated 89 kDa PARP cleavage. Atypical PARP cleavage in CaPcells after β-lap exposure correlated well with apoptosis (Table 3) andclonogenic lethality, which was abrogated by dicoumarol. Atypical PARPcleavage was not affected by administration of 100 μM zVAD-fink, aglobal caspase inhibitor, or DVED-fmk (Table 3). To date, no knowncaspases are activated in NQO1 expressing CaP or breast cancer cellsafter β-lap treatments (Tagliarino). Interestingly, lamin B cleavage(proteolysis observed during apoptosis) was noted in NQO1⁺DU-145 cellsafter β-lap treatments. Cleavage of lamin B (60 kDa) to a 46 kDapolypeptide aids in nuclear matrix breakdown during apoptosis (Rao, L.,Perez, D. and White, E. (1996) Journal of Cell Biology 135, 1441-1455).The pan-caspase inhibitor, 100 μM zVAD-fmk, did not inhibit lamin Bcleavage after β-lap (Pink, J. J., Wuerzberger-Davis, S., Tagliarino,C., Planchon, S. M., Yang, X., Froelich, C. J. and Boothman, D. A.(2000) Exp Cell Res 255, 144-155). In mutant p53-expressing DU-145cells, β-lap exposure resulted in p53 cleavage (40 and ˜20 kDa) thatwere not inhibited by 100 μM zVAD-fmk. A similar p53 cleavage wasdescribed during μ-calpain-mediated apoptosis of neural cells (Kubbutat,M. H. and Vousden, K. H. (1997) Mol Cell Biol 17, 460-468; Shinohara,K., Tomioka, M., Nakano, H., Tone, S., Ito, H. and Kawashima, S. (1996)Biochem J 317, 385-388; Vanags, D. M., Orrenius, S. andAguilar-Santelises, M. (1997) Br J Haematol 99, 824-831).

Expression of NQO1 sensitizes LNCaP cells to β-lap alone. LNCaP cellswere transfected with either pcDNA3 empty vector or pcDNA3 containingfull-length NQO1 cDNA. Five NQO1-containing (LN-NQ Cl 1-4, 10) and onevector alone (LN-pcDNA3) LNCaP clones were isolated (FIG. 14) and didnot differ in P450 or b5R activities. In clonogenic assays,NQO1-deficient parental LNCaP cells were more resistant to β-lap thanDU-145 or PC-3 cells. After transfection, NQO1-containing LNCaP cloneswere significantly more sensitive to β-lap than LNCaP cells containingpcDNA3 vector alone (FIG. 14), and as sensitive to β-lap as PC-3 orDU-145 cells. Dicoumarol administration returned NQO1-expressing LNCaPclones to the relative β-lap-resistant phenotype seen with LNCaPparental cells. Interestingly, NQO1-containing LN-NQ Cl 10 cells wereresistant to menadione compared to NQO1-deficient LN-pcDNA3 cells.Dicournarol reversed this resistance (FIG. 14B). NQO1-expressing LNCaPclone (LN-NQ Cl 1-4, 10) exposed to 10 μM β-lap resulted in significantapoptosis (i.e., 80-90%) compared to LNCaP vector alone clones, whichshowed <5% apoptosis. Dicoumarol prevented β-lap-induced apoptosis andatypical PAkP cleavage in NQO1⁺LNCaP clones. Similar results were shownin breast cancer cell lines.

NQO1 is an IR-inducible gene and is necessary for radiosensitization. Wefound that NQO1 was induced in U1-Mel cells, which are dramaticallyradiosensitized by β-lap. Treatment of U1-Mel cells with IR resulted ina dramatic increase in NQO1 transcript levels, cloned as xip3 (Boothman,D. A., Meyers, M., Fukunaga, N. and Lee, S. W. (1993) Proceedings of theNational Academy of Science of the United States of America 90,7200-7204). Peak levels of NQO1 were found 4-8 h post-IR. NQO1 levelswere induced by as little as 1.0 Gy, a clinical dose of IR. Due to thelimited availability of CaP cell lines expressing low NQO1 levels,studies on induction of NQO1 in CaP cells have been limited. All celllines available to us have elevated endogenous NQO1 levels and wereradiosensitized by β-lap. NQO1⁻LNCaP cells were not sensitized.

NQO1 is necessary for radiosensitization, but IR-induction is notrequired. To answer the question of whether NQO1 levels must be inducedfor β-lap radiosensitization, we examined NQO1⁺LNCaP NQCl2 v.NQO1⁻LNCaP-pcDNA3 vector alone cells for differences inradiosensitization by various concentrations of β-lap, given 4 h post-IR(FIG. 15). Dose response clonogenic survival assays revealed two basicconclusions. First, only NQO1-expressing LNCaP cells wereradiosensitized by β-lap treatments (FIG. 155B). Second, expression ofNQO1 alone did not confer radioprotection of cells; it was possible thatIR-induction of NQO1 would confer a survival advantage of cellsfollowing IR exposures. Finally, if the dose of β-lap was too high(i.e., 5 μM, FIG. 15B), radiosensitization was abolished due to thecytotoxicity of β-lap alone. Similar results were found in breast cancercell lines expressing or lacking NQO1. Thus, NQO1 expression wasnecessary for radiosensitization, and induction of the enzyme is notrequired for enhanced cell killing of IR-exposed cells by β-lap. This isimportant since NQO1-overexpressing CaP tumors should be sensitized byβ-lap whether they induce, or already express, this enzyme; CaP tumorshave 5- to 10-fold higher NQO1 levels.

β-lap bioavailability and antitumor activity. To show efficacy of β-lapin vivo, a systemic delivery system was developed for thiswater-insoluble drug. β-Lap complexes withβ-hydroxypropyl-β-cyclodextrin (HP-β-CD) that dissolves the drug andmakes it bioavailable. The toxicity of HP-β-CD.β-lap in 18-20 gm athymicnude mice was examined and the maximum tolerated dose (MTD) was ˜75mg/kg. This represents an ˜3-fold greater bioavailability of β-lap invivo than in previous studies using lipiodol, where an LD₅₀ of >150mg/kg was noted for β-lap in C57/blk6 mice, and antitumor activity equalto taxol was noted with human ovarian xenografts. We tested the effectsof β-lap administered i.p. in athymic nude mice bearing NQO1⁺ MDA-MB-468(468) human breast cancer xenografts. Significant antitumor activity wasnoted in athymic mice bearing 468 xenografts when β-lap was administeredat 55-70 mg/kg (FIG. 16).

REFERENCES

All publications and patents mentioned herein, are hereby incorporatedby reference in their entirety as if each individual publication orpatent was specifically and individually indicated to be incorporated byreference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-28. (canceled)
 29. A drug delivery composition selected from animplant, microparticles, and nanoparticles, wherein the compositioncomprises a prodrug of a lapachone incorporated in a biocompatiblepolymer having a molecular weight of about 2,000 or more daltons, andthe lapachone has a structure of Formula I or II:

wherein R and R₁ each independently represent h, hydroxy, amino, amido,sulfhydryl, halogen, or substituted or unsubstituted alkyl, alkenyl,heteroalkyl, carbocyclic aliphatic, carbocyclic aliphatic alkyl, aryl,aralkyl, heterocyclic aliphatic, heterocyclic aliphatic alkyl,heteroaryl, heteroaralkyl, or alkoxy, or a pharmaceutically acceptablesalt thereof:
 30. The composition of claim 29, wherein the prodrug ofthe lapachone is admixed with the polymer.
 31. The composition of claim29, wherein the composition is an implant.
 32. The composition of claim31, wherein the implant is a millirod dimensioned to position tworadiation seeds at a predetermined distance from one another.
 33. Thecomposition of claim 29, wherein the composition is provided asmicroparticles.
 34. The composition of claim 29, wherein the compositionis provided as nanoparticles.
 35. The composition of claim 29, furthercomprising hydroxypropyl β-cyclodextrin, wherein the prodrug of thelapachone is provided as an inclusion complex with the hydroxypropylβ-cyclodextrin.
 36. The composition of claim 29, wherein the prodrug oftie lapachone is a prodrug of β-lapachone.
 37. The composition of claim29, wherein the polymer is biodegradable.
 38. The composition of claim29, wherein the polymer comprises one or more of poly(lactic-co-glycolicacid) (PLGA), poly(lactic acid) (PLA), polyethylene glycol (PEG),polysebacic acid (PSA), or a polyanhydride.
 39. A method of inhibitingproliferation of a cancerous cell in a patient, comprising administeringto the patient the drug delivery composition of claim
 29. 40. The methodof claim 39, wherein the cell overexpresses NQO1.
 41. The method ofclaim 40, wherein the cell is a lung cancer cell, a breast cancer cell,or a prostate cancer cell.
 42. The method of claim 41, wherein the cellis a non-small cell lung cancer cell.
 43. The method of claim 42,wherein the composition is delivered to the patient by inhalation ofmicro spheres comprising a pro drug of a lapachone and a biocompatiblepolymer.
 44. The method of claim 39, wherein the cell is a prostatecancer cell and the composition is delivered to the patient byimplanting radioactive seeds spaced apart by at least one polymericmillirod comprising a prodrug of a lapachone and a biocompatiblepolymer.
 45. A composition of claim 29, wherein a therapeutic agent, adiagnostic agent, an imaging agent, or an adjuvant is also incorporatedin the polymer.
 46. A kit comprising a prodrug of a lapachone, aβ-cyclodextrin, and instructions for combining the prodrug of thelapachone and β-cyclodextrin to form a complex and administering thecomplex to a patient.
 47. A kit comprising at least two radioactiveseeds, at least one millirod according to claim 4, and instructions foradministering the radioactive seeds and millirod to a patient.
 48. Acomposition of claim 29, wherein the biocompatible polymer has amolecular weight of about 2,000 to about 1,000,000 daltons.